Isoxazole Compounds As Inhibitors Of Heat Shock Proteins

ABSTRACT

Isoxazoles of formula (A) or (B) are inhibitors of HSP90 activity, and useful for treatment of, for example cancers: 
     
       
         
         
             
             
         
       
     
     wherein R 1 , is a group of formula (IA): —Ar 1 -(Alk 1 ) p -(Z) r -(Alk 2 ) s -Q, wherein in any compatible combination Ar 1  is an optionally substituted aryl or heteroaryl radical, Alk 1  and Alk 2  are optionally substituted divalent C 1 -C 6  alkylene or C 2 -C 6  alkenylene radicals, p, r and s are independently 0 or 1, Z is -0-, —S—, —(C═O)—, —(C═S)—, —SO.sub.2-, —C(═O)O—, —C(═O)NR A —, —C(═S)NR A —, —SO 2 NR A —, —NR A C(═O)—, —NR A SO 2 — or —NR A — wherein R A  is hydrogen or C 1 -C 6  alkyl, and Q is hydrogen or an optionally substituted carbocyclic or heterocyclic radical; R 2  is (i) a group of formula (IA) above or (ii) a carboxamide radical; or (iii) a non aromatic carbocyclic or heterocyclic ring wherein a ring carbon is optionally substituted, and/or a ring nitrogen is optionally substituted by a group of formula -(Alk 1 )p-(Z) r -(Alk 2 ) s -Q wherein Q, Alk 1 , Alk 2 , Z, p, r and s are as defined above in relation to group (IA); and R 3  is hydrogen, optionally substituted cycloalkyl, cycloalkenyl, C 1 -C 6  alkyl, C 1 -C 6  alkenyl, or C 1 -C 6  alkynyl; or a carboxyl, carboxamide, or carboxyl ester group.

This invention relates to substituted isoxazoles having HSP90 inhibitoryactivity, to the use of such compounds in medicine, in relation todiseases which are responsive to inhibition of HSP90 activity such ascancers, and to pharmaceutical compositions containing such compounds.

BACKGROUND TO THE INVENTION

Molecular chaperones maintain the appropriate folding and conformationof proteins and are crucial in regulating the balance between proteinsynthesis and degradation. They have been shown to be important inregulating many important cellular functions, such as cell proliferationand apoptosis (Jolly and Morimoto, 2000; Smith et al., 1998; Smith,2001).

Heat Shock Proteins (HSPs)

Exposure of cells to a number of environmental stresses, including heatshock, alcohols, heavy metals and oxidative stress, results in thecellular accumulation of a number of chaperones, commonly known as heatshock proteins (HSPs). Induction of HSPs protects the cell against theinitial stress insult, enhances recovery and leads to maintenance of astress tolerant state. It has also become clear, however, that certainHSPs may also play a major molecular chaperone role under normal,stress-free conditions by regulating the correct folding, degradation,localization and function of a growing list of important cellularproteins.

A number of multigene families of HSPs exist, with individual geneproducts varying in cellular expression, function and localization. Theyare classified according to molecular weight, e.g., HSP70, HSP90, andHSP27.

Several diseases in humans can be acquired as a result of proteinmisfolding (reviewed in Tytell et al., 2001; Smith et al., 1998). Hencethe development of therapies which disrupt the molecular chaperonemachinery may prove to be beneficial. In some conditions (e.g.,Alzheimer's disease, prion diseases and Huntington's disease), misfoldedproteins can cause protein aggregation resulting in neurodegenerativedisorders. Also, misfolded proteins may result in loss of wild typeprotein function, leading to deregulated molecular and physiologicalfunctions in the cell.

HSPs have also been implicated in cancer. For example, there is evidenceof differential expression of HSPs which may relate to the stage oftumour progression (Martin et al., 2000; Conroy et al., 1996; Kawanishiet al., 1999; Jameel et al., 1992; Hoang et al., 2000; Lebeau et al.,1991). As a result of the involvement of HSP90 in various criticaloncogenic pathways and the discovery that certain natural products withanticancer activity are targeting this molecular chaperone, thefascinating new concept has been developed that inhibiting HSP functionmay be useful in the treatment of cancer. The first molecular chaperoneinhibitor is currently undergoing clinical trials.

HSP90

HSP90 constitutes about 1-2% of total cellular protein, and is usuallypresent in the cell as a dimer in association with one of a number ofother proteins (see, e.g., Pratt, 1997). It is essential for cellviability and it exhibits dual chaperone functions (Young et al., 2001).It plays a key role in the cellular stress response by interacting withmany proteins after their native conformation has been altered byvarious environmental stresses, such as heat shock, ensuring adequateprotein folding and preventing non-specific aggregation (Smith et al.,1998). In addition, recent results suggest that HSP90 may also play arole in buffering against the effects of mutation, presumably bycorrecting the inappropriate folding of mutant proteins (Rutherford andLindquist, 1998). However, HSP90 also has an important regulatory role.Under normal physiological conditions, together with its endoplasmicreticulum homologue GRP94, HSP90 plays a housekeeping role in the cell,maintaining the conformational stability and maturation of several keyclient proteins. These can be subdivided into three groups: (a) steroidhormone receptors, (b) Ser/Thr or tyrosine kinases (e.g., ERBB2, RAF-1,CDK4, and LCK), and (c) a collection of apparently unrelated proteins,e.g., mutant p53 and the catalytic subunit of telomerase hTERT. All ofthese proteins play key regulatory roles in many physiological andbiochemical processes in the cell. New HSP90 client proteins arecontinuously being identified.

The highly conserved HSP90 family in humans consists of four genes,namely the cytosolic HSP90α and HSP90β isoforms (Hickey et al., 1989),GRP94 in the endoplasmic reticulum (Argon et al., 1999) and HSP75/TRAP1in the mitochondrial matrix (Felts et al., 2000). It is thought that allthe family members have a similar mode of action, but bind to differentclient proteins depending on their localization within the cell. Forexample, ERBB2 is known to be a specific client protein of GRP94 (Argonet al., 1999) and type 1 tumour necrosis factor receptor (TNFR1) and RBhave both been shown to be clients of TRAP1 (Song et al., 1995; Chen etal., 1996).

HSP90 participates in a series of complex interactions with a range ofclient and regulatory proteins (Smith, 2001). Although the precisemolecular details remain to be elucidated, biochemical and X-raycrystallographic studies (Prodromou et al., 1997; Stebbins et al., 1997)carried out over the last few years have provided increasingly detailedinsights into the chaperone function of HSP90.

Following earlier controversy on this issue, it is now clear that HSP90is an ATP-dependent molecular chaperone (Prodromou et al, 1997), withdimerization of the nucleotide binding domains being essential for ATPhydrolysis, which is in turn essential for chaperone function (Prodromouet al, 2000a). Binding of ATP results in the formation of a toroidaldimer structure in which the N terminal domains are brought into closercontact with each other resulting in a conformational switch known asthe ‘clamp mechanism’ (Prodromou and Pearl, 2000b).

Known HSP90 Inhibitors

The first class of HSP90 inhibitors to be discovered was thebenzoquinone ansamycin class, which includes the compounds herbimycin Aand geldanamycin. They were shown to reverse the malignant phenotype offibroblasts transformed by the v-Src oncogene (Uehara et al., 1985), andsubsequently to exhibit potent antitumour activity in both in vitro(Schulte et al., 1998) and in vivo animal models (Supko et al., 1995).

Immunoprecipitation and affinity matrix studies have shown that themajor mechanism of action of geldanamycin involves binding to HSP90(Whitesell et al., 1994; Schulte and Neckers, 1998). Moreover, X-raycrystallographic studies have shown that geldanamycin competes at theATP binding site and inhibits the intrinsic ATPase activity of HSP90(Prodromou et al., 1997; Panaretou et al., 1998). This in turn preventsthe formation of mature multimeric HSP90 complexes capable ofchaperoning client proteins. As a result, the client proteins aretargeted for degradation via the ubiquitin proteasome pathway.17-Allylamino, 17-demethoxygeldanamycin (17AAG) retains the property ofHSP90 inhibition resulting in client protein depletion and antitumouractivity in cell culture and xenograft models (Schulte et al, 1998;Kelland et al, 1999), but has significantly less hepatotoxicity thangeldanamycin (Page et al, 1997). 17AAG is currently being evaluated inPhase I clinical trials.

Radicicol is a macrocyclic antibiotic shown to reverse the malignantphenotype of v-Src and v-Ha-Ras transformed fibroblasts (Kwon et al,1992; Zhao et al, 1995). It was shown to degrade a number of signallingproteins as a consequence of HSP90 inhibition (Schulte et al., 1998).X-ray crystallographic data confirmed that radicicol also binds to the Nterminal domain of HSP90 and inhibits the intrinsic ATPase activity (Roeet al., 1998). Radicicol lacks antitumour activity in vivo due to theunstable chemical nature of the compound.

Coumarin antibiotics are known to bind to bacterial DNA gyrase at an ATPbinding site homologous to that of the HSP90. The coumarin, novobiocin,was shown to bind to the carboxy terminus of HSP90, i.e., at a differentsite to that occupied by the benzoquinone ansamycins and radicicol whichbind at the N-terminus (Marcu et al., 2000b). However, this stillresulted in inhibition of HSP90 function and degradation of a number ofHSP90-chaperoned signalling proteins (Marcu et al., 2000a). Geldanamcyincannot bind HSP90 subsequent to novobiocin; this suggests that someinteraction between the N and C terminal domains must exist and isconsistent with the view that both sites are important for HSP90chaperone properties.

A purine-based HSP90 inhibitor, PU3, has been shown to result in thedegradation of signalling molecules, including ERBB2, and to cause cellcycle arrest and differentiation in breast cancer cells (Chiosis et al.,2001).

HSP90 as a Therapeutic Target

Due to its involvement in regulating a number of signalling pathwaysthat are crucially important in driving the phenotype of a tumour, andthe discovery that certain bioactive natural products exert theireffects via HSP90 activity, the molecular chaperone HSP90 is currentlybeing assessed as a new target for anticancer drug development (Neckerset al., 1999).

The predominant mechanism of action of geldanamycin, 17AAG, andradicicol involves binding to HSP90 at the ATP binding site located inthe N-terminal domain of the protein, leading to inhibition of theintrinsic ATPase activity of HSP90 (see, e.g., Prodromou et al., 1997;Stebbins et al., 1997; Panaretou et al., 1998).

Inhibition of HSP90 ATPase activity prevents recruitment ofco-chaperones and encourages the formation of a type of HSP90heterocomplex from which these client proteins are targeted fordegradation via the ubiquitin proteasome pathway (see, e.g., Neckers etal., 1999; Kelland et al., 1999).

Treatment with HSP90 inhibitors leads to selective degradation ofimportant proteins involved in cell proliferation, cell cycle regulationand apoptosis, processes which are fundamentally important in cancer.

Inhibition of HSP90 function has been shown to cause selectivedegradation of important signalling proteins involved in cellproliferation, cell cycle regulation and apoptosis, processes which arefundamentally important and which are commonly deregulated in cancer(see, e.g., Hostein et al., 2001). An attractive rationale fordeveloping drugs against this target for use in the clinic is that bysimultaneously depleting proteins associated with the transformedphenotype, one may obtain a strong antitumour effect and achieve atherapeutic advantage against cancer versus normal cells. These eventsdownstream of HSP90 inhibition are believed to be responsible for theantitumour activity of HSP90 inhibitors in cell culture and animalmodels (see, e.g., Schulte et al., 1998; Kelland et al., 1999).

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the use of a class of substitutedisoxazole compounds as HSP90 inhibitors; for example for inhibition ofcancer cell proliferation. The invention also includes novel isoxazolecompounds per se, and pharmaceutical compositions containing them

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention there is provided the use of acompound of formula (A) or (B) or a salt, N-oxide, hydrate or solvatethereof, or a prodrug thereof, in the preparation of a composition forinhibition of HSP90 activity:

wherein

-   R₁ is a group of formula (IA):

—Ar¹-(Alk¹)_(p)-(Z)_(r)-(Alk²)_(s)-Q  (IA)

wherein in any compatible combination

-   -   Ar¹ is an optionally substituted aryl or heteroaryl radical,    -   Alk¹ and Alk² are optionally substituted divalent C₁-C₆ alkylene        or C₂-C₆ alkenylene radicals,    -   p, r and s are independently 0 or 1,    -   Z is —O—, —S—, —(C═O)—, —(C═S)—, —SO₂—, —C(═O)O—, —C(═O)NR^(A)—,        —C(═S)NR^(A)—, —SO₂NR^(A)—, —NR^(A)C(═O)—, —NR^(A)SO₂— or        —NR^(A)— wherein R^(A) is hydrogen or C₁-C₆ alkyl, and    -   Q is hydrogen or an optionally substituted carbocyclic or        heterocyclic radical;        R₂ is (i) a group of formula (IA) as defined in relation to R₁;    -   (ii) a carboxamide radical; or    -   (iii) a non aromatic carbocyclic or heterocyclic ring wherein a        ring carbon is optionally substituted, and/or a ring nitrogen is        optionally substituted by a group of formula        -(Alk¹)_(p)-(Z)_(r)-(Alk²)_(s)-Q wherein Q, Alk¹, Alk², Z, p, r        and s are as defined above in relation to group (IA); and        R₃ is hydrogen, optionally substituted cycloalkyl, cycloalkenyl,        C₁-C₆ alkyl, C₁-C₆ alkenyl, or C₁-C₆ alkynyl; or a carboxyl,        carboxamide, or carboxyl ester group.

In general, the class of compounds defined above in relation to formula(I) is believed to be novel, and the invention includes all novelmembers of that class and their salts, hydrates and solvates, andprodrugs thereof.

As used herein:

-   -   the term “carboxyl group” refers to a group of formula —COOH;    -   the term “carboxyl ester group” refers to a group of formula        —COOR, wherein R is a radical actually or notionally derived        from the hydroxyl compound ROH; and    -   the term “carboxamide group” refers to a group of formula        —CONR_(a)R_(b), wherein —NR_(a)R_(b) is a primary or secondary        (including cyclic) amino group actually or notionally derived        from ammonia or the amine HNR_(a)R_(b).

As used herein, the term “(C_(a)-C_(b))alkyl” wherein a and b areintegers refers to a straight or branched chain alkyl radical havingfrom a to b carbon atoms. Thus when a is 1 and b is 6, for example, theterm includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term “divalent (C_(a)-C_(b))alkylene radical” whereina and b are integers refers to a saturated hydrocarbon chain having froma to b carbon atoms and two unsatisfied valences.

As used herein, the term “(C_(a)-C_(b))alkenyl” wherein a and b areintegers refers to a straight or branched chain alkenyl radical havingfrom a to b carbon atoms and containing at least one double bond of E orZ configuration, including for example, ethenyl and allyl.

As used herein the term “divalent (C_(a)-C_(b))alkenylene radical”wherein a and b are integers refers to a hydrocarbon chain having from ato b carbon atoms, at least one double bond, and two unsatisfiedvalences.

As used herein, the term “(C_(a)-C_(b))alkynyl” wherein a and b areintegers refers to a straight or branched chain alkenyl radical havingfrom a to b carbon atoms and containing at least one triple bond,including for example, ethynyl and prop-2-ynyl.

As used herein, the term “divalent (C_(a)-C_(b))alkynylene radical”wherein a and b are integers refers to a straight or branched chainalkynyl radical having from a to b carbon atoms and containing at leastone triple bond, and two unsatisfied valencies.

As used herein the term “cycloalkyl” refers to a saturated carbocyclicradical having from 3-8 carbon atoms and includes, for example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl.

As used herein the term “cycloalkenyl” refers to a carbocyclic radicalhaving from 3-8 carbon atoms containing at least one double bond, andincludes, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl andcyclooctenyl.

As used herein the term “aryl” refers to a mono-, bi- or tri-cycliccarbocyclic aromatic radical. Illustrative of such radicals are phenyl,biphenyl and napthyl.

As used herein the term “carbocyclic” refers to a cyclic radical whosering atoms are all carbon, and includes monocyclic aryl, cycloalkyl andcycloalkenyl radicals.

As used herein the term “heteroaryl” refers to a mono-, bi- ortri-cyclic aromatic radical containing one or more heteroatoms selectedfrom S, N and O. Illustrative of such radicals are thienyl, benzthienyl,furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl,benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl,benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl,benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic”includes “heteroaryl” as defined above, and in particular means a mono-,bi- or tri-cyclic non-aromatic radical containing one or moreheteroatoms selected from S, N and O, and to groups consisting of amonocyclic non-aromatic radical containing one or more such heteroatomswhich is covalently linked to another such radical or to a monocycliccarbocyclic radical. Illustrative of such radicals are pyrrolyl,furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl,thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl,pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl,benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl,ethylenedioxyphenyl, maleimido and succinimido groups.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with upto four compatible substituents, each of which independently may be, forexample, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy, hydroxy(C₁-C₆)alkyl,mercapto, mercapto(C₁-C₆)alkyl, (C₁-C₆)alkylthio, halo (includingfluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro,nitrile (—CN), oxo, phenyl, —COOH, —COOR^(A), —COR^(A), —SO₂R^(A),—CONH₂, —SO₂NH₂, —CONHR^(A), —SO₂NHR^(A), —CONR^(A)R^(B),—SO₂NR^(A)R^(B), —NH₂, —NHR^(A), —NR^(A)R^(B), —OCONH₂, —OCONHR^(A),—OCONRAR^(B), —NHCOR^(A), —NHCOOR^(A), —NR^(B)COOR^(A), —NHSO₂OR^(A),—NR^(B)SO₂OH, —NR^(B)SO₂OR^(A), —NHCONH₂, —NR^(A)CONH₂, —NHCONHR^(B),—NR^(A)CONHR^(B), —NHCONR^(A)R^(B), or —NR^(A)CONR^(A)R^(B) whereinR^(A) and R^(B) are independently a (C₁-C₆)alkyl group. An “optionalsubstituent” may be one of the foregoing substituent groups. Of theabove substituents, (C₁-C₆)alkyl, halo, trifluoromethyl,trifluoromethoxy, trifluoromethylsulfonyl, and phenyl are those mostcommonly regarded as lipophilic. Other substituents listed which containalkyl groups may be lipophilic depending on the particular alkyl groupspresent.

As used herein the term “salt” includes base addition, acid addition andquaternary salts. Compounds of the invention which are acidic can formsalts, including pharmaceutically or veterinarily acceptable salts, withbases such as alkali metal hydroxides, e.g. sodium and potassiumhydroxides; alkaline earth metal hydroxides e.g. calcium, barium andmagnesium hydroxides; with organic bases e.g. N-methyl-D-glucamine,choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethylpiperidine, dibenzylamine and the like. Those compounds (1) which arebasic can form salts, including pharmaceutically or veterinarilyacceptable salts with inorganic acids, e.g. with hydrohalic acids suchas hydrochloric or hydrobromic acids, sulphuric acid, nitric acid orphosphoric acid and the like, and with organic acids e.g. with acetic,tartaric, succinic, fumaric, maleic, malic, salicylic, citric,methanesulphonic, p-toluenesulphonic, be4nzoic, benzenesunfonic,glutamic, lactic, and mandelic acids and the like.

The term “lipophilic” as used herein in relation to a substituent meansthat it has a positive substituent hydrophobicity constant (π). (Apositive value for it indicates that the substituent is more lipophilicthan hydrogen, whereas a negative value indicates it is less lipophilic,i.e. more hydrophilic, than hydrogen).

Some compounds of the invention contain one or more actual or potentialchiral centres because of the presence of asymmetric carbon atoms. Thepresence of several asymmetric carbon atoms gives rise to a number ofdiastereoisomers with R or S stereochemistry at each chiral centre. Theinvention includes all such diastereoisomers and mixtures thereof.

An aspect of the invention includes compounds of formula (A) or (B)above and a salts, N-oxides, hydrates or solvates thereof and prodrugsthereof, except the following three compounds (X), (Y) and (Z) which arecommercially available:

Subject to those exclusions, the invention particularly includes thosewherein the substituents R₁, R₂ and R₃ are as discussed and specified inthe following sections headed “The radical R₁”, “The radical R₂”, and“The radical R₃”, Another aspect includes the use of such compounds forthe treatment of diseases responsive to inhibition of HSP90 activity.

The Radical R₁

In general, it is currently preferred that the radical Ar¹ present inthe R₁ group is optionally substituted phenyl, preferably with one ofthe optional substituents being a hydroxy group in position 2 relativeto the point of attachment of the phenyl ring to isoxazole ring. Inother words, the group R₁ preferably has formula (IB)

wherein Alk¹, Alk², p, r, s, Z and Q are as defined above in relation toR₁, and R represents one or more optional substituents. In suchstructures, it is further preferred that the ring carbon atom adjacentthe hydroxyl group be unsubstituted. In the further discussion of R₁which follows, this preference applies in addition to any otherpossibilities mentioned.

In the simplest structures with which the invention is concerned, eachof p, r and s may be 0, and Q may be hydrogen, so that R₁ is optionallysubstituted aryl or heteroaryl. In such cases, R₁ may be, for example,optionally substituted phenyl, preferably 2-hydroxyphenyl which may befurther substituted, for example by one or more of hydroxy, methyl,ethyl, methoxy, ethoxy, chloro, or bromo. Currently preferred arecompounds wherein R₁ is 2,4-dihydroxyphenyl, substituted in the5-position by a small lipophilic substituent, for example having amolecular volume equal to or less than that of tert-butyl, such asmethyl, ethyl, isopropyl, isobutyl, tert-butyl, chloro, or bromo,especially ethyl, isopropyl, or chloro. In such 5-substituted,2,4-diyhdroxy phenyl compounds of the invention, the hydroxyl groups maybe protected by groups which are cleaved in the body to release thehydroxyl groups. Known prodrug-type groups of this kind which arecleaved to hydroxyls include alkylcarbonyloxy groups such asmethylcarbonyloxy, and alkylaminocarbonyloxy groups such asdialkylamino- or isopropylamino-carbonyloxy.

In other simple structures with which the invention is concerned, p, rand s may again each be 0, and Q may be an optionally substitutedcarbocyclic or heterocyclic ring, for example a phenyl or pyridyl ring.In such cases, Q is a direct substituent in the optionally substitutedAr¹ ring

In more complex structures with which the invention is concerned, one ormore of p, r and s may be 1, and Q may be hydrogen or an optionallysubstituted carbocyclic or heterocyclic ring. For example, p and/or smay be 1 and r may be 0, so that Q is linked to Ar¹ by an alkylene oralkenylene radical, for example a C₁-C₃ alkylene radical, which isoptionally substituted. In other cases each of p, r, and s may be 1, inwhich cases, Q is linked to Ar¹ by an alkylene or alkenylene radicalwhich is interrupted by the hetero atom-containing Z radical. In stillother cases, p and s may be 0 and r may be 1, in which case Q is linkedto Ar¹ via the hetero atom-containing Z radical.

Specific examples of R₁ groups of the above types are present in thecompounds of the Examples herein.

The Radical R₂

When R₂ is of type (i), i.e. a group of formula (IA), examples includephenyl, 2-, 3-, or 4-pyridyl, 2- or 3-furanyl, 2- or 3-thienyl, andthiazolyl wherein optional substituents include any of those listedabove in the definition of “substituted”, for example methoxy, ethoxy,methylenedioxy, ethylenedioxy, fluoro, chloro, bromo, andtrifluoromethyl. For example R₂ may be phenyl substituted in the 4position by C₁-C₆ alkoxy such as methoxy or ethoxy, or by fluoro,chloro, bromo, piperazinyl, N-methylpiperazinyl, or piperidinyl.

Presently preferred R₂ substituents include those having the partialstructure:

wherein the substituted amino group —NR¹⁰R¹¹ is a solubilising group.Many such solubilising groups are known in medicinal chemistry. Examplesinclude morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, ethylamino,isopropylamino, diethylamino, cyclohexylamino, cyclopentylamino,methoxyethylamino, piperidin-4-yl, N-acetylpiperazinyl,methylsulfonylamino, thiomorpholinyl, thiomorpholinyldioxide,4-hydroxyethylpiperidinyl, and 4-hydroxypiperidinyl.

Our copending international patent application no. PCT/GB2003/005275discloses HSP90 inhibiting pyrazole compounds analogous to theisoxazoles with which this invention is concerned, and which arebelieved to bind to the HSP90 target in an analogous fashion. Thosepyrazole compounds have a carboxamide group in the positioncorresponding to R₂ of the present isoxazoles. Hence, when R₂ in thepresent isoxazoles is a carboxamide radical of type (ii) above, examplesinclude those present in the pyrazole compounds of PCT/GB2003/005275,for example carboxamides of formula —CONR^(B)(Alk)_(n)R^(A) wherein

-   -   Alk is a divalent alkylene, alkenylene or alkynylene radical,        for example a —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH═CH—, or        —CH₂CCCH₂-radical, and the Alk radical may be optionally        substituted,    -   n is 0 or 1,    -   R^(B) is hydrogen or a C₁-C₆ alkyl or C₂-C₆ alkenyl group, for        example methyl, ethyl, n- or iso-propyl, or allyl,    -   R^(A) is hydroxy or optionally substituted carbocyclic, for        example hydroxy and/or chloro-substituted phenyl and 3,4        methylenedioxyphenyl; or heterocyclyl, for example pyridyl,        furyl, thienyl, N-piperazinyl, or N-morpholinyl any of which        heterocyclic rings may be substituted,    -   or R^(A) and R^(B) taken together with the nitrogen to which        they are attached form an N-heterocyclic ring which may        optionally contain one or more additional hetero atoms selected        from O, S and N, and which may optionally be substituted on one        or more ring C or N atoms, examples of such N-heterocyclic rings        including morpholino, piperidinyl, piperazinyl and        N-phenylpiperazinyl.

The Radical R₃

R₃ may be, for example, hydrogen, methyl, ethyl, n- or iso-propyl,trifluoromethyl, hydroxyethyl, methylsulfonaminomethyl, or a carboxamidegroup —CONR^(B)(Alk)_(n)R^(A) as discussed above for R₂. A carboxamidegroup is presently preferred, especially ethylaminocarbonyl andisopropylaminocarbonyl.

A particular sub-set of the compounds with which this invention isconcerned consists of those of formula (ID), and the formula Bregioisomers thereof, and their salts, solvates and hydrates, andprodrugs thereof:

wherein each R independently represents an optional substituent and R³represents a carboxamide group.

A preferred sub-set of the compounds with which this invention isconcerned consists of those of formula (IE), and the formula (B)regioisomers thereof, and their salts, solvates and hydrates, andprodrugs thereof:

wherein R₃ represents a carboxamide group (such as ethylaminocarbonylCH₃CH₂NHC(═O)—, or isopropylaminocarbonyl (CH₃)₂CHNHC(═O)—); R₉represents —CH₂NR¹⁰R¹¹ or —NR¹⁰R¹¹ wherein the substituted amino group—NR¹⁰R¹¹ is a solubilising group, (such as morpholinyl, piperidinyl,piperazinyl, pyrrolidinyl, ethylamino, isopropylamino, diethylamino,cyclohexylamino, cyclopentylamino, methoxyethylamino, piperidin-4-yl,N-acetylpiperazinyl, N-methylpiperazinyl, methylsulfonylamino,thiomorpholinyl, thiomorpholinyl-dioxide, 4-hydroxyethylpiperidinyl, and4-hydroxypiperidinyl); and R₈ represents an optional substituent,especially a small lipophilic group (such as ethyl, isopropyl, bromo, orchloro).

Specific compounds with which the invention is concerned include thoseof the Examples, particularly the following, and their salts, N-oxides,hydrates and solvates, and prodrugs thereof:

-   5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-piperidin-1-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   4-(4-Diethylaminomethyl-phenyl)-5-(2,4-dihydroxy-5-isopropyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-[4-(4-methyl-piperazin-1-ylmethyl)-phenyl]-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-ethylaminomethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Di    hydroxy-5-isopropyl-phenyl)-4-[4-(isopropylamino-methyl)-phenyl]-isoxazole-3-carboxylic    acid ethylamide-   4-(4-Cyclohexylaminomethyl-phenyl)-5-(2,4-dihydroxy-5-isopropyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   4-[4-(tert-Butylamino-methyl)-phenyl]-5-(2,4-dihydroxy-5-isopropyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-{4-[(2-methoxy-ethylamino)-methyl]-phenyl}-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid isopropylamide-   5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-[4-(4-methyl-piperazin-1-ylmethyl)-phenyl]-isoxazole-3-carboxylic    acid isopropylamide-   5-(5-tert-Butyl-2,4-dihydroxy-phenyl)-4-[4-(4-methyl-piperazin-1-ylmethyl)-phenyl]-isoxazole-3-carboxylic    acid ethylamide-   5-(5-tert-Butyl-2,4-dihydroxy-phenyl)-4-(4-piperidin-1-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Dihydroxy-5-isobutyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Dihydroxy-5-isobutyl-phenyl)-4-(4-piperidin-1-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(5-tert-Butyl-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethyl amide-   5-(5-tert-Butyl-2,4-dihydroxy-phenyl)-4-(4-diethylaminomethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   3-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-5-carboxylic    acid ethylamide-   4-(4-Diethylaminomethyl-phenyl)-5-(4,6-dihydroxy-2′-methy-biphenyl-3-yl)-isoxazole-3-carboxylic    acid ethylamide-   4-(4-Diethylaminomethyl-phenyl)-5-(4′-fluoro-4,6-dihydroxy-biphenyl-3-yl)-isoxazole-3-carboxylic    acid ethylamide-   4-(4-Diethylaminomethyl-phenyl)-5-(4,6-dihydroxy-biphenyl-3-yl)    isoxazole-3-carboxylic acid ethylamide-   5-(2′-Fluoro-4,6-dihydroxy-biphenyl-3-yl)-4-(4-pyrrolidin-1-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(4,6-Dihydroxy-biphenyl-3-yl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(2,4-Dihydroxy-5-phenethyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-piperidin-1-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid isopropylamide-   4-(4-Diethylaminomethyl-phenyl)-5-(5-ethyl-2,4-dihydroxy-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(5-Ethyl-2,4-dihydroxy-phenyl)-4-[4-(4-methyl-piperazin-1-ylmethyl)-phenyl]-isoxazole-3-carboxylic    acid ethylamide-   5-(5-Ethyl-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-diethylaminomethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide-   5-(5-Chloro-2,4-dihydroxy-phenyl)-4-[4-(4-methyl-piperazin-1-ylmethyl)-phenyl]-isoxazole-3-carboxylic    acid ethylamide-   5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylic    acid ethylamide

Compounds with which the invention is concerned may be prepared byliterature methods, such as those of the preparative Examples herein,and methods analogous thereto.

For example, some compounds of formula (IA) may be prepared by reactionof hydroxylamine and a compound of formula (III)

wherein ring A corresponds to the group R₁ of compounds (IA) and R₂ andR₃ are as defined in relation to formula (I). Compounds prepared in thisway may then be chemically modified to introduce desired substituents,to produce other compounds of formula (A) For example where R₁ is aphenyl ring, optionally already carrying substituents, the introductionof a bromo substituent will often enable introduction of othersubstituents at the bromo site by sp2 coupling.

In another route to some compounds of formula (A), the isoxazole ring isformed by the reaction of a compound (IV) with hydroxylamine

wherein R′₁ and R′₃ are members of the substituent classes R₁ and R₃defined above, to produce the isoxazole (V)

followed by introduction of the additional substituent R₂ (for exampleby bromination or iodination of the ring carbon in (V) and sp2 coupling,and/or modification of the resultant R¹ ₁, R¹ ₃ and R₂ substituents ofthe isoxazole.

Furthermore, some isoxazole regioisomers (B) may be prepared from theisoxazoles (A) by reaction with trimethyloxonium boron trifluoride, andagain compounds prepared in this way may then be chemically modified tointroduce desired substituents, to produce other compounds of formula(IA).

It will be understood that during the above syntheses, it may bedesirable to protect any reactive groups such as hydroxyls, and todeprotect later. Further synthetic details are described in the examplesherein.

The compounds of the invention are inhibitors of HSP90 and are thususeful in the treatment of diseases which are responsive to inhibitionof HSP90 activity such as cancers; viral diseases such as Hepatitis C(HCV) (Waxman, 2002); Immunosupression such as in transplantation(Bijlmakers, 2000 and Yorgin, 2000); Anti-inflammatory diseases (Bucci,2000) such as Rheumatoid arthritis, Asthma, MS, Type I Diabetes, Lupus,Psoriasis and Inflammatory Bowel Disease; Cystic fibrosis (Fuller,2000); Angiogenesis-related diseases (Hur, 2002 and Kurebayashi, 2001):diabetic retinopathy, haemangiomas, psoriasis, endometriosis and tumourangiogenesis. Also an Hsp90 inhibitor of the invention may protectnormal cells against chemotherapy-induced toxicity and be useful indiseases where failure to undergo apoptosis is an underlying factor.Such an Hsp90 inhibitor may also be useful in diseases where theinduction of a cell stress or heat shock protein response could bebeneficial, for example, protection from hypoxia-ischemic injury due toelevation of Hsp70 in the heart (Hutter, 1996 and Trost, 1998) and brain(Plumier, 1997 and Rajder, 2000). An Hsp90 inhibitor could also beuseful in diseases where protein misfolding or aggregation is a majorcausal factor, for example, scrapie/CJD, Huntingdon's and Alzheimer's(Sittler, 2001; Trazelt, 1995 and Winklhofer, 2001).

Accordingly, the invention also provides:

(i) a method of treatment of diseases or conditions responsive toinhibition of HSP90 activity in mammals, particularly humans, whichmethod comprises administering to the mammal an amount of a compound offormula (A) or (B) as defined above, or a salt, hydrate or solvatethereof, effective to inhibit said HSP90 activity; and(ii) a compound of formula (A) or (B) as defined above, or a salthydrate or solvate thereof, for use in human or veterinary medicine,particularly in the treatment of diseases or conditions responsive toinhibition of HSP90 activity;(iii) a pharmaceutical composition comprising a compound of formula (A)or (B) as defined and specified above, together with a pharmaceuticallyacceptable carrier. In particular, the invention includes a solution orsuspension of such compound in a sterile, physiologically acceptablecarrier, for example aqueous saline.

It will be understood that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination and the causative mechanism and severity ofthe particular disease undergoing therapy. In general, a suitable dosefor orally administrable formulations will usually be in the range of0.1 to 3000 mg once, twice or three times per day, or the equivalentdaily amount administered by infusion or other routes. However, optimumdose levels and frequency of dosing will be determined by clinicaltrials as is conventional in the art.

The compounds with which the invention is concerned may be prepared foradministration by any route consistent with their pharmacokineticproperties. The orally administrable compositions may be in the form oftablets, capsules, powders, granules, lozenges, liquid or gelpreparations, such as oral, topical, or sterile parenteral solutions orsuspensions. Tablets and capsules for oral administration may be in unitdose presentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art,for example as described in standard textbooks of pharmaceutics such asthe British Pharmacopoeia.

The active ingredient may also be administered parenterally in a sterilemedium. Depending on the vehicle and concentration used, the drug caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants, such as a local anaesthetic, preservative and bufferingagents, can be dissolved in the vehicle.

Compounds of the invention are also useful in in vitro assays dependenton inhibition of HSP90 activity, for example in screening foralternative classes of HSP90 inhibitors wherein the test compoundcompetes with or displaces a compound of this invention. Accordingly, inyet another aspect, the invention includes a method of inhibiting HSP90activity, comprising bringing into contact, in vitro, an HSP90 enzymeand a compound of formula (A) or (B) as defined and specified above.

The following examples illustrate the preparation and activities ofspecific compounds of the invention.

Examples 1-4

Example 14-[4-(4-Methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol

Step 1 1-(2,4-Dihydroxy-phenyl)-2-(4-methoxy-phenyl)-ethanone

Resorcinol (4.4 g, 40 mmol) and 4-methoxyphenylacetic acid (6.6 g, 40mmol) in boron trifluoride.etherate (25 ml, 0.2 mol) was heated, under anitrogen atmosphere, at 90° C. for ˜90 mins. to give a pale redsolution. The solution was allowed to cool and poured into aqueoussodium acetate (200 ml, 10%) and the mixture stirred to give a paleyellow precipitate. The solids were removed by filtration and washedwith water (200 ml). Solids were taken up in ethyl acetate (250 ml) andwashed with water (200 ml). Solution was dried over anhyrous magnesiumsulphate and concentrated, to a yellow semi-solid. Trituration withdiethyl ether (100 ml) gave the1-(2,4-dihydroxy-phenyl)-2-(4-methoxy-phenyl)-ethanone as a pale orangesolid, dried in vacuo, (2.2 g)

LC retention time 2.39 minutes [M+H]⁺ 259.2 (Run time 3.75 mins)

N.M.R (DMSO-d₆) 7.95 (d J 8.9 Hz ArH) 7.2 (d J 8.7 Hz 2ArH) 6.9 (d J 8.7Hz 2ArH) 6.4 (d J 9.9 ArH) 6.25 (s ArH) 4.2 (s 2CH₂) 3.75 (s 3OCH₃)

Step 2 7-Hydroxy-3-(4-methoxy-phenyl)-2-methyl-chromen-4-one

Acetic anhydride (3 ml, 30 mmol) was added to a suspension of potassiumcarbonate (4.0 g, 29 mmol) and1-(2,4-dihydroxy-phenyl)-2-(4-methoxy-phenyl)-ethanone (1.95 g, 7.5mmol) in DMF (10 ml), and the resulting suspension heated at 115° C. for˜90 mins. The mixture was allowed to cool and poured into water (200ml), to give an off-white precipitate. The solids were removed byfiltration and washed with water (100 ml) and diethyl ether (2×40 ml),to give 7-hydroxy-3-(4-methoxy-phenyl)-2-methyl-chromen-4-one as anoff-white powder, dried in vacuo, (1.65 g)

LC retention time 2.26 minutes [M+H]⁺ 283.2 (Run time 3.75 mins)

N.M.R (DMSO-d₆) 7.8 (d J 8.7 Hz ArH) 7.2 (d J 8.8 Hz 2ArH) 7.0 (d J 8.8Hz 2ArH) 6.9 (d J 8.7 ArH) 6.8 (s ArH) 3.8 (s 3OCH₃) 2.2 (s 3CH₃)

Step 3 4-[4-(4-Methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol

Hydroxylamine hydrochloride (0.35 g, 5 mmol) was added to a suspensionof 7-hydroxy-3-(4-methoxy-phenyl)-2-methyl-chromen-4-one (0.14 g, 0.5mmol) in pyridine (3 ml) and the mixture heated under reflux for ˜4 hrs.The solution was allowed to cool and poured into water (50 ml) andextracted with diethyl ether (50 ml). The extracts were washed withwater (3×50 ml) and saturated aqueous sodium chloride solution (30 ml).The solution was dried over anhydrous magnesium sulphate andconcentrated to give a pale brown gum.

Crude product was purified by column chromatography, on silica, elutingwith ethyl acetate/hexane (1:2), to give a colourless gum. Triturationwith hexane gave4-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol as awhite powder, dried in vacuo, (0.087 g)

LC retention time 2.20 minutes [M+H]⁺ 298.2 (Run time 3.75 mins)

N.M.R (DMSO-d₆) 7.1 (d J 8.8 Hz 2ArH) 6.85 (d J 8.6 Hz ArH) 6.8 (d J 8.8Hz 2ArH) 6.25 (s ArH) 6.15 (d J 8.6 Hz ArH) 3.65 (s 3OCH₃) 2.15 (s 3CH₃)

Example 24-Bromo-6-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol

Benzyltrimethylammonium tribromide (3.95 g, 10 mmol) was addedportion-wise to an ice cooled suspension of4-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol(Example 1) (2.95 g, 10 mmol) in dichloromethane (50 ml) and the mixturestirred for ˜60 mins, at room temperature. Ethyl acetate (300 ml) wasadded and the mixture washed with water (3×200 ml) and saturated aqueoussodium chloride solution (50 ml). The solution was dried over anhydrousmagnesium sulphate and concentrated to give a pale brown solid. Crudeproduct was purified by column chromatography, on silica, eluting withethyl acetate/hexane (1:2), to give4-bromo-6-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diolas a white solid, dried in vacuo, (3.42 g)

LC retention time 2.38 minutes [M+H]⁺ 378.2 (Run time 3.75 mins)

N.M.R (Acetone-d₆) 7.35 (s ArH) 7.2 (d J 8.8 Hz 2ArH) 6.9 (d J 8.8 Hz2ArH) 6.65 (s ArH) 3.8 (s 3OCH₃) 2.25 (s 3CH₃)

Example 35-[4-(4-Methoxy-phenyl)-3-methyl-isoxazol-5-yl]-biphenyl-2,4-diol

Step 15-(2,4-Bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-3-methyl-isoxazole

Benzyl bromide (0.36 ml, 3 mmol) was added suspension of4-bromo-6-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol(Example 2) (0.55 g, 1.5 mmol) and cesium carbonate (0.85 g, 2.6 mmol)in DMF (5 ml) and the mixture stirred for ˜18 hrs, at room temperature.Water (100 ml) was added and the mixture extracted with diethyl ether(2×30 ml). The combined extracts were washed with water (4×75 ml) andsaturated aqueous sodium chloride solution (50 ml). The solution wasdried over anhydrous magnesium sulphate and concentrated to give a palebrown gum. Trituration with hexane gave5-(2,4-bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-3-methyl-isoxazoleas an off-white solid, dried in vacuo, (0.5 g).

LC retention time 3.08 minutes [M+H]⁺ 558.4 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.55 (s ArH) 7.35-7.25 (m 5ArH) 7.2 (m 3ArH) 6.95(d J 8.8 Hz 2ArH) 6.85 (m 2ArH) 6.7 (d J 8.8 Hz 2ArH) 6.35 (s ArH) 4.95(s 2CH₂) 4.6 (s 2CH₂) 3.75 (s 3OCH₃) 2.25 (s 3CH₃)

Step 25-(4,6-Bis-benzyloxy-biphenyl-3-yl)-4-(4-methoxy-phenyl)-3-methyl-isoxazole

Potassium phosphate (0.1 g, 0.5 mmol) was added to a solution of5-(2,4-bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-3-methyl-isoxazole(0.14 g, 0.25 mmol) and phenyl boronic acid (0.095 g, 0.75 mmol) in 1,4dioxan (4 ml) under a nitrogen atmosphere.Tetrakis(triphenylphosphine)palladium(0) (cat.) was added and thesuspension heated, 80° C. for ˜18 hrs. The suspension was allowed tocool and ethyl acetate (25 ml) added. The mixture was washed with water(3×25 ml) and saturated aqueous sodium chloride solution (25 ml). Thesolution was dried over anhydrous magnesium sulphate and concentrated togive a pale brown gum. Trituration with hexane gave5-(4,6-bis-benzyloxy-biphenyl-3-yl)-4-(4-methoxy-phenyl)-3-methyl-isoxazoleas an off-white solid, dried in vacuo.

LC retention time 3.08 minutes [M+H]⁺ 554.4 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.4 (m 2ArH) 7.35 (s ArH) 7.3-7.1 (m 11ArH) 6.95 (dJ 8.8 Hz 2ArH) 6.9 (m 2ArH) 6.7 (d J 8.8 Hz 2ArH) 6.45 (s ArH) 4.9 (s2CH₂) 4.7 (s 2CH₂) 3.75 (s 3OCH₃) 2.25 (s 3CH₃)

Step 3 7-Hydroxy-3-(4-methoxy-phenyl)-2-methyl-6-phenyl-chromen-4-one

Ammonium formate (3.2 g, 50 mmol) was added to a solution of5-(4,6-bis-benzyloxy-biphenyl-3-yl)-4-(4-methoxy-phenyl)-3-methyl-isoxazole(1.4 g, 2.5 mmol) in methanol (20 ml)/ethyl acetate (10 ml) under anitrogen atmosphere. Palladium on carbon (10%) (cat.) was added and thesuspension heated, at 60° C. for ˜18 hrs. The suspension was allowed tocool and ethyl acetate (150 ml) added, and the suspension filtered. Thefiltrate was washed with water (3×100 ml) and saturated aqueous sodiumchloride solution (50 ml). The solution was dried over anhydrousmagnesium sulphate and concentrated to give a pale brown gum.Trituration with methanol gave7-hydroxy-3-(4-methoxy-phenyl)-2-methyl-6-phenyl-chromen-4-one as anoff-white solid, dried in vacuo.

LC retention time 2.58 minutes [M+H]⁺ 359.2 (Run time 3.75 mins)

N.M.R (DMSO-d₆) 7.9 (s ArH) 7.5-7.3 (m 5ArH) 7.25 (d J 8.8 Hz 2ArH) 7.1(s ArH) 7.05 (d J 8.8 Hz 2ArH) 3.85 (s 3OCH₃) 2.2 (s 3CH₃)

Step 4 5-[4-(4-Methoxy-phenyl)-3-methyl-isoxazol-5-yl]-biphenyl-2,4-diol

Hydroxylamine hydrochloride (75 mg, 1.08 mmol) was added to a suspensionof 7-hydroxy-3-(4-methoxy-phenyl)-2-methyl-6-phenyl-chromen-4-one (105mg, 0.29 mmol) in pyridine (2 ml) and the mixture heated under refluxfor ˜6 hrs., to give a pale yellow solution. The solution was allowed tocool and water (20 ml) added. The mixture was extracted with diethylether (2×10 ml). The combined extracts were washed with water (2×20 ml)and saturated aqueous sodium chloride solution (10 ml). The solution wasdried over anhydrous magnesium sulphate and concentrated. The crudeproducts were purified by column chromatography, silica, eluting withethyl acetate/hexane (1:1), to give the title compound as an off-whitepowder (80 mg)

LC retention time 2.56 minutes [M+H]⁺ 374.3 (Run time 3.75 mins)

N.M.R (Acetone-d₆) 7.5-7.3 (m 5ArH) 7.2 (d J 8.8 Hz 2ArH) 7.0 (d J 8.8Hz 2ArH) 6.9 (d J 8.6 Hz ArH) 6.35 (s ArH) 6.1 (d J 8.7 Hz ArH) 3.85 (s3OCH₃) 2.25 (s 3CH₃)

Example 44-Chloro-6-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol

Hydroxylamine hydrochloride (0.7 g, 10 mmol) was added to a suspensionof 6-chloro-7-hydroxy-3-(4-methoxy-phenyl)-2-methyl-chromen-4-one[prepared analogously to Example 1, Step 2] (0.32 g, 1.0 mmol) inpyridine (4 ml) and the mixture heated under reflux for ˜6 hrs., to givea pale yellow solution. The solution was allowed to cool and water (20ml) added. The mixture was extracted with diethyl ether (2×10 ml). Thecombined extracts were washed with water (2×20 ml) and saturated aqueoussodium chloride solution (10 ml). The solution was dried over anhydrousmagnesium sulphate and concentrated. The crude products were purified bycolumn chromatography, silica, eluting with ethyl acetate/hexane (1:1),to give4-chloro-6-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diolas an off-white powder (0.103 g)

LC retention time 2.37 minutes [M+H]⁺ 332.2 (Run time 3.75 mins)

N.M.R (Acetone-d₆) 7.2 (d J 8.8 Hz 2ArH) 7.15 (s ArH) 6.9 (d J 8.8 Hz2ArH) 6.6 (s ArH) 3.85 (s 3OCH₃) 2.25 (s 3CH₃)

The compounds of Examples 1-4 had an HSP90 IC50 in the range A whentested in the Malachite Green ATPase assay described below. In thefollowing tables, the final column gives the result on the same basisfor the compound in question, except in the case of Example 12b, wherethe activity quoted is as measured in the fluorescence polarisationassay described below.

Examples 5-16 were prepared using the reaction described for Examples1-4. Other details of the preparation Examples 6 and 7 are analagous tothose of Examples 86 and 87.

Hsp90 Example Structure MH+ IC50  5* 

326 B  6  

330 B  7  

296 B  8  

349 B  9  

286 A  10   

303 A  11   

342 A  12   

375 B  12a  

367 A  12b**

323 A***  12c^(§) 

351 A  12d^(§) 

343 A *Also available commercially from Interbioscreen ^(§)availablecommercially from Enamine **prepared from protected bromo resorcinolintermediate with copper (I) cyanide in dimethylformamide at 150° C.***Fluorescence Polarisation Assay: ‘A’ = <10 uM; ‘B’ = >10 uM

Example 144-[4-(4-Methoxy-phenyl)-3-methyl-isoxazol-5-yl]-6-phenethyl-benzene-1,3-diol

was prepared from styryl boronic acid coupling of the bromo isoxazolecompound of Example 2 Step 1, as described above, followed by reductionand treatment with hydroxylamine, analogously to Example 3.

LC retention time 2.56 minutes [M+H]⁺ 402 (Run time 3.75 mins)

Example 154-[4-(4-Methoxy-phenyl)-3-methyl-isoxazol-5-yl]-2,6-bis-(4-methyl-piperazin-1-ylmethyl)-benzene-1,3-diol

N-methylpiperazine (0.125 ml, 1.1 mmol) was added to a suspension of4-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol (0.15g, 0.5 mmol) and paraformaldehyde (0.040 g) in 1,4-dioxan (4 ml) and themixture heated under reflux for ˜18 hrs., to give a brown yellowsolution. The solution was allowed to cool and ethyl acetate (25 ml)added. The mixture was washed with water (3×25 ml) and saturated aqueoussodium chloride solution (25 ml). The solution was dried over anhydrousmagnesium sulphate and concentrated to a pale brown gum. Triturationwith hexane, gave4-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-2,6-bis-(4-methyl-piperazin-1-ylmethyl)-benzene-1,3-diol(0.121 g) as a pale brown powder.

LC retention time 1.61 minutes [M+H]⁺ 522.6 (Run time 3.75 mins)

N.M.R (Acetone-d₆) 7.2 (d J 8.8 Hz 2ArH) 6.95 (s ArH) 6.8 (d J 8.8 Hz2ArH) 3.85 (s 3OCH₃) 3.75 (s 2CH₂) 3.65 (s 2CH₂) 2.9-2.0 (br s 16 CH₂)2.3 (s 3CH₃) 2.25 (s 3CH₃) 2.2 (s 3CH₃)

Example 162,4-Dihydroxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzoicacid methyl ester

Step 1

n-Butyl lithium (100 μl) was added to a solution of5-(2,4-bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-3-methyl-isoxazole(154 mg, 0.28 mmol) in tetrahydrofuran (2.5 ml) under a nitrogenatmosphere at −78° C. Solution stirred at −70° C. for 30 minutes to givean orange solution. The ion was quenched with methyl chloroformate (100μl, 3 eq) and allowed to warm to room temperature for 30 minutes. Thesolution was quenched with saturated aqueous ammonium chloride (5 ml).The mixture was extracted with ethyl acetate (3×5 ml). The combinedextracts were washed with water (2×5 ml) and saturated aqueous sodiumchloride solution (5 ml). The solution was dried over anhydrousmagnesium sulphate and concentrated. The crude products were purified bycolumn chromatography, silica, eluting with ethyl acetate in hexane(gradient 20% to 60% ethyl acetate) to give2,4-Bis-benzyloxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzoicacid methyl ester (72 mg).

LC retention time 4.95 minutes [M+H]⁺ 536.4 (Run time 7.5 mins)

N.M.R (DMSO-de) 7.8 (s ArH) 7.55 (d J 7.1 Hz 2ArH) 7.4 (t J 6.2 Hz 2ArH)7.35 (d J 6.1 Hz ArH) 7.3 (m 3ArH) 7.1 (m 4ArH) 7.0 (s ArH) 6.9 (d 8.8Hz 2ArH) 5.3 (s 2CH₂) 5.1 (s 2CH₂) 3.78 (s OCH₃) 3.76 (s OCH₃) 2.28 (sCH₃)

Step 2

Ammonium formate (172 mg, 20 eq) was added to a solution of2,4-Bis-benzyloxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzoicacid methyl ester (72 mg, 0.13 mmol) in methanol (2 ml)/ethyl acetate (1ml) under a nitrogen atmosphere. 10% Palladium on carbon (cat.) wasadded and the suspension heated at 60° C. overnight. The solution wasallowed to cool. Ethyl acetate (5 ml) added, solution washed with water(2×5 ml) and saturated aqueous sodium chloride solution (5 ml). Thesolution was dried over anhydrous magnesium sulphate and concentrated.The crude products were purified by column chromatography, silica,eluting with ethyl acetate in hexane (gradient 25% to 45% ethyl acetate)to give2,4-dihydroxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzoicacid methyl ester (7.0 mg).

LC retention time 2.49 minutes [M+H]⁺ 356.3 (Run time 3.75 mins)

N.M.R (CDCl₃) δ=10.85 (s ArOH) 7.52 (s ArOH) 7.12 (d J8 Hz 2ArH) 6.98 (sArH) 6.91 (d J8 Hz 2ArH) 6.45 (s ArH) 3.78 (s 3 OCH₃) 3.71 (s 3 OCH₃)2.21 (s 3 CH₃).

The compounds of Examples 14-16 had an HSP90 IC50 in the ranges ‘A’, ‘B’and ‘B’, respectively when tested in the Malachite Green ATPase assaydescribed below.

Similarly, Examples 17-20 were prepared quenching with N-formylpiperidine, phenyl thioisocyanate, 2-methoxy phenyl isocyanate andbenzaldehyde, respectively. The final deprotection reaction was carriedout with boron trichloride as described for example 23 (last reaction onScheme 5). Example 21 was a by-product from Step 1, Example 16. Quotedactivities are those obtained in the Malachite Green Assay describedbelow.

Hsp90 Example Structure MH+ IC50 17

326 B 18

433 B 19

447 A 20

418 A 21

354 A

Example 224-Benzyl-6-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-benzene-1,3-diol

Carbonic acid 2-benzoyl-5-ethoxycarbonyloxy-phenyl ester ethyl ester

Triethyl amine (10 ml, 72.2 mmol) was added to a solution of2,4-dihydroxybenzophenone (1) (5.4 g, 23.3 mmol) in THF (50 ml) and thesolution cooled to 0° C. Ethyl chloroformate (6.9 ml, 72.2 mmol) wasadded slowly and the suspension stirred for ˜30 mins at 0° C., and for˜3 hrs at room temperature. Water (150 ml) was added and the mixtureextracted with diethyl ether (150 ml). The extracts were washed withwater (2×150 ml) and saturated aqueous sodium chloride solution (100ml). The solution was dried over anhydrous magnesium sulphate andconcentrated to give 4-benzyl-benzene-1,3-diol as a pale green gum,solidified on standing, (8.2 g).

LC retention time 2.73 minutes [M+H]⁺ 359.2 (Run time 3.75 mins)

δ (Chloroform-d) 7.7 (m 2ArH) 7.5 (m 2ArH) 7.35 (m 2ArH) 7.15 (m 2ArH)4.25 (q J 7.1 Hz 2CH₂) 4.05 (q J 7.1 Hz 2 CH₂) 1.35 (t J 7.1 Hz 3CH₃)1.15 (t J 7.1 Hz 3 CH₃)

4-benzyl-benzene-1,3-diol

A solution of sodium borohydride (1.85 g, 49 mmol) in water (30 ml) wasadded to an ice cooled solution of carbonic acid2-benzoyl-5-ethoxycarbonyloxy-phenyl ester ethyl ester (3.6 g, 10 mmol)in THF (30 ml). The mixture was stirred for ˜60 mins. at 0° C., and for˜60 hrs. at room temperature, to give a pale red suspension. Water (150ml) was added and the mixture extracted with diethyl ether (150 ml). Theextracts were washed with water (2×100 ml) and saturated aqueous sodiumchloride solution (50 ml). The solution was dried over anhydrousmagnesium sulphate and concentrated to give a pale yellow gum. The gumwas taken up in aqueous sodium hydroxide (20 ml, 10%), and the solutionheated under reflux for ˜60 mins. The solution was allowed to cool andacidified with hydochloric acid (5 ml, 37%). The mixture was extractedwith diethyl ether (50 ml). The extracts were washed with water (3×40ml) and saturated aqueous sodium chloride solution (30 ml). The solutionwas dried over anhydrous magnesium sulphate and concentrated to give4-benzyl-benzene-1,3-diol as a dark red gum, (2.1 g).

LC retention time 2.28 minutes [M+H]⁺ no ion (Run time 3.75 mins) δ(Chloroform-d) 7.2 (m 3ArH) 7.1 (m 2ArH) 6.85 (d J 8.1 Hz ArH) 6.3 (d J8.1 Hz ArH) 6.2 (s ArH) 3.85 (s 2CH₂)

The 4-benzyl-benzene-1,3-diol was used as the starting material in aScheme 1 synthesis to provide Example 23.

Example 233-{2,4-Dihydroxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-phenyl}-acrylicacid

Step 13-{2,4-Bis-benzyloxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-phenyl}-acrylicacid tert-butyl ester

Diisopropylethyl amine (1 ml, 5.7 mmol) was added to a suspension of5-(2,4-Bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-3-methyl-isoxazole(0.56 g, 1.0 mmol) in tert-butyl acrylate (1 ml, 6.8 mmol) and 1-butanol(8 ml) under a nitrogen atmosphere.Dichlorobis(tri-o-tolylphosphine)palladium (II) (cat.) was added and thesuspension heated, 140° C. for ˜18 hrs., to give a yellow/greensolution. The solution was allowed to cool and concentrated to ayellow/green gum. The crude product was purified by columnchromatography, silica, eluting with ethyl acetate/hexane (1:9), to give3-{2,4-Bis-benzyloxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-phenyl}-acrylicacid tert-butyl ester as a yellow/green gum (315 mg). Starting material(170 mg) was recovered.

LC retention time 3.23 minutes [M+H]⁺ 604.6 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.85 (d J 16.1 Hz CH) 7.6 (s ArH) 7.4-7.25 (m 8ArH)7.05 (d J 8.8 Hz 2ArH) 6.9 (m 2ArH) 6.8 (d J 8.8 Hz 2ArH) 6.5 (s ArH)6.35 (d J 16.1 Hz CH) 5.05 (s 2CH₂) 4.75 (s 2CH₂) 3.75 (s 3OCH₃) 2.25 (s3CH₃) 1.5 (s 9CCH₃)

Step 23-{2,4-Dihydroxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-phenyl}-acrylicacid

Boron trichloride solution (2 ml, 1.0M in dichloromethane) was addedslowly to a solution of3-{2,4-Bis-benzyloxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-phenyl}-acrylicacid tert-butyl ester (50 mg, 0.09 mmol) in dichloromethane (1 ml), at−78° C. (dry ice/acetone) under a nitrogen atmosphere. The resultingsolution was stirred for ˜1 hr at −78° C., and for ˜90 mins. at roomtemperature. The solution was cooled to −78° C. and water (2 ml) addedand the mixture was stirred for ˜30 mins at room temperature. Ethylacetate (30 ml) was added and the solution washed with water (2×5 ml)and saturated aqueous sodium chloride solution (10 ml). The solution wasdried over anhydrous magnesium sulphate and concentrated to a paleyellow gum. Trituration with hexane gave a yellow solid, solids wereremoved by filtration and washed with hexane, dried in vacuo, to give3-{2,4-dihydroxy-5-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-phenyl}-acrylicacid (10 mg) as yellow powder.

LC retention time 2.08 minutes [M+H]⁺ 368.3 (Run time 3.75 mins)

N.M.R (Acetone-d₆) 7.85 (d J 16.1 Hz CH) 7.5 (s ArH) 7.25 (d J 8.8 Hz2ArH) 6.95 (d J 8.8 Hz 2ArH) 6.6 (s ArH) 6.35 (d J 16.1 Hz CH) 3.8 (s3OCH₃) 2.25 (s 3CH₃)

Similarly,4-[4-(4-methoxy-phenyl)-3-methyl-isoxazol-5-yl]-6-styryl-benzene-1,3-diol(Example 24) was prepared by boron trichloride deprotection of5-(2,4-Bis-benzyloxy-5-styryl-phenyl)-4-(4-methoxy-phenyl)-3-methyl-isoxazole(prepared from styryl boronic acid coupling of bromo isoxazoleintermediate, Example 3))

LC retention time 2.08 minutes [M+H]⁺ 368.3 (Run time 3.75 mins)

The compounds of Examples 22-24 had an HSP90 IC50 in the ranges ‘A’, ‘B’and ‘C’ respectively when tested in the Malachite Green ATPase assaydescribed below.

Example 255-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxy-phenyl)-isoxazole-3-carboxylicacid ethylamide

Step 1 1-(5-Chloro-2,4-dihydroxy-phenyl)-ethanone

Acetic acid (17.5 mL) was added dropwise to a suspension of4-chlororesorcinol (42.5 g, 0.293 mmol) in boron trifluoride etherate(200 mL) under a nitrogen atmosphere. The reaction mixture was heated at90° C. for 3.5 hours and then allowed to cool to room temperature. Asolid had formed after around 1 hour of cooling. The mixture was pouredinto 700 mL of a 10% w/v aqueous sodium acetate solution. This mixturewas stirred vigorously for 2.5 hours. A light brown solid had formedwhich was filtered, washed with water and air-dried overnight to afford1-(5-chloro-2,4-dihydroxy-phenyl)-ethanone (31.6 g, 58%). LCMS: [M−H]⁺185.

Step 2 1-(2,4-Bis-benzyloxy-5-chloro-phenyl)-ethanone

Benzyl bromide (30 mL) was added to a mixture of1-(5-chloro-2,4-dihydroxy-phenyl)-ethanone (20 g, 0.107 moles) andpotassium carbonate (37 g, 2.5 equiv) in acetonitrile (350 mL). Themixture was heated at reflux for 6 hours then allowed to cool andstirred overnight. The mixture was filtered and the solids were washedwith dichloromethane (3×100 mL). The combined organic extracts wereevaporated in vacuo to leave a pale yellow solid which was trituratedwith a mixture of hexane (350 mL)/ethyl acetate (15 mL) and filtered togive an off-white solid, 1-(2,4-bis-benzyloxy-5-chloro-phenyl)-ethanone(35.4 g, 90%). 1H NMR (400 MHz) consistent with structure.

Step 3 4-(2,4-bis-benzyloxy-5-chlorophenyl)-2-hydroxy-4-oxo-but-2-enoicacid ethyl ester

Sodium metal (1.35 g, 0.058 mol) was added in small pieces over a periodof 20 minutes to stirred anhydrous ethanol under a nitrogen atmosphere.The reaction mixture was then stirred for a further 10 minutes until allthe sodium had reacted to give a homogeneous solution.1-(2,4-bis-benzyloxy-5-chloro-phenyl)-ethanone (10.0 g, 0.027 mol) wasadded in portions over 2-3 minutes and the resulting suspension wasstirred for 5 minutes prior to addition of diethyl oxalate (6 ml, 0.043mol) which afforded a thicker, yellow precipitate. The reaction mixturewas heated to reflux (giving homogeneous brown solution) for 4 hours,then allowed to cool to room temperature and acetic acid (6 ml) wasadded. The resulting which solid forms was triturated, filtered, washedwith ethanol and dried to give a yellow solid (12.0 g, 95%). ¹H NMR (400MHz, CDCl₃) δ 1.2 (t, 3H), 4.19 (q, 2H), 5.05 (s, 2H), 5.10 (s, 2H),6.50 (s, 1H), 7.22-7.41 (m, 10H), 7.97 (s, 1H).

Step 4 5-(2,4-Bis-benzyloxy-5-chlorophenyl)-isoxazole-3-carboxylic acidethyl ester

Hydroxylamine hydrochloride (0.89 g; 12.8 mmol) was added to asuspension of4-(2,4-bis-benzyloxy-5-chlorophenyl)-2-hydroxy-4-oxo-but-2-enoic acidethyl ester (5.00 g; 10.7 mmol) in absolute ethanol (100 ml). Thereaction mixture was heated at reflux for four hours then allowed tocool to ambient temperature (during this time the mixture remainsheterogeneous but becomes lighter yellow in colour). The mixture wasfiltered and the filtered solid was washed with water (2×20 ml), ethanol(2×20 ml) and dried in vacuo at 45° C. This affords5-(2,4-bis-benzyloxy-5-chlorophenyl)-isoxazole-3-carboxylic acid ethylester as a fluffy yellow solid, 4.49 g (91%) LCMS: [M+H]⁺ 466, 464(³⁷Cl; ³⁵Cl).). ¹H NMR (400 MHz, CDCl₃) 1.42 (t, 3H), 4.42 (q, 2H), 5.13(s, 2H), 5.14 (s, 2H), 6.62 (s, 1H), 7.01 (s, 1H), 7.35-7.43 (m, 10H),8.00 (s, 1H).

Step 5 5-(2,4-Bis-benzyloxy-5-chlorophenyl)-isoxazole-3-carboxylic acidethylamide

A solution of ethylamine in methanol solution (2.0M; 40 mL; 80 mmol) wasadded to a stirred suspension of5-(2,4-bis-benzyloxy-5-chlorophenyl)-isoxazole-3-carboxylic acid ethylester (4.40 g; 9.51 mmol) in absolute ethanol (50 ml). The reactionmixture was heated to 80° C. (oil-bath temperature) for five hours. Thereaction mixture was allowed to cool to ambient temperature and left tostand overnight. A colourless solid product formed and the reactionmixture was further cooled in an ice-water bath, filtered and washedwith cold ethanol (2×20 ml). The colourless product was dried in vacuoto afford 5-(2,4-bis-benzyloxy-5-chlorophenyl)-isoxazole-3-carboxylicacid ethylamide 3.42 g (78%) LCMS: [M+H]⁺ 465, 463 (³⁷Cl; ³⁵Cl). ¹H NMR(400 MHz, CDCl₃) δ 1.25 (t, 3H), 3.48 (m, 2H), 5.10 (s, 2H), 5.2 (s,2H), 6.59 (s, 1H), 6.83 (brt, 1H), 7.08 (s, 1H), 7.30-7.41 (m, 10H),7.97 (s, 1H).

Step 65-(2,4-Bis-benzyloxy-5-chlorophenyl)-4-bromo-isoxazole-3-carboxylic acidethylamide

A solution of bromine in acetic acid (0.6M; 7.2 mL; 4.32 mmol) was addedto a stirred suspension of5-(2,4-Bis-benzyloxy-5-chlorophenyl)-4-bromo-isoxazole-3-carboxylic acidethylamide (2.00 g; 4.32 mmol) and sodium acetate (0.708 g, 8.64 mmol)in acetic acid (30 ml) at ambient temperature. The mixture was heated to80° C. and becomes homogeneous within 5-10 minutes, to afford a dark redsolution. After heating for 2.5 hours the solution was yellow in colour.TLC analysis showed starting material and product present. A further 2.0ml (1.2 mmol) of the bromine in acetic acid solution was added over thenext two hours. The reaction mixture was allowed to cool to ambienttemperature and acetic acid was removed in vacuo to afford a solidresidue, which was partitioned between ether (200 ml) and water (200ml). The phases were separated and the organic phase was washed withwater (3×100 ml), saturated aqueous sodium bicarbonate solution (2×100ml) and saturated sodium chloride solution (1×200 ml). The organic phasewas dried over sodium sulphate, filtered and the filtrate solvents wereremoved in vacuo to afford a yellow oil which was purified by flashchromatography on silica gel, eluting with 1-20% ethyl acetate inhexane. This affords product as colourless solid, 1.2 g (52%) LCMS:[M+H]⁺ 543, 541 (⁸¹Br; ⁷⁹Br). ¹H NMR (400 MHz, CDCl₃) δ 1.26 (t, 1H),3.50 (m, 2H), 5.01 (s, 2H), 5.12 (s, 2H), 6.62 (s, 1H), 6.74 (br t, 1H),2.28-7.41 (m, 10H), 7.53 (s, 1H).

Step 75-(2,4-Bis-benzyloxy-5-chlorophenyl)-4-(4-methoxy-phenyl)-isoxazole-3-carboxylicacid ethylamide

To a mixture of 4-methoxyphenylboronic acid (0.178 g, 1.17 mmol) and5-(2,4-Bis-benzyloxy-5-chlorophenyl)-4-bromo-isoxazole-3-carboxylic acidethylamide (0.507 g, 0.94 mmol) was added sodium hydrogen carbonate (237mg, 2.82 mmol) followed by DMF (5 mL) and water (1.0 mL). The mixturewas degassed by evacuation and flushing with nitrogen (three times),followed by bubbling nitrogen gas through mixture for five minutes.Dichlorobis(triphenylphosphine)palladium (II) (66 mg, 0.094 mmol) wasadded and reaction mixture was heated under a nitrogen atmosphere at 90°C. for two hours (reaction mixture becomes dark brown in colour).Another 10 mg of dichlorobis(triphenylphosphine)palladium (II) was addedand reaction mixture was heated at 90° C. for 15 hours then allowed tocool to ambient temperature. The majority of solvents were removed invacuo and the residue was partitioned between ethyl acetate (50 mL) andwater (50 mL). This mixture was filtered through a pad of celite toremove palladium residues and then the phases were separated and theorganic phase was washed with water (2×30 mL), saturated aqueous sodiumchloride solution (50 mL) then dried over sodium sulphate. The mixturewas filtered and the filtrate solvents were removed in vacuo to afford ayellow oil (598 mg). The crude reaction product was purified byadsorption onto silica gel then flash chromatography on silica gel (20 gIST) eluting with a solvent gradient of 1 to 20% ethyl acetate inhexane. This affords5-(2,4-Bis-benzyloxy-5-chlorophenyl)-4-(4-methoxy-phenyl)-isoxazole-3-carboxylicacid ethylamide as a colourless solid (0.223 g, 40%). LCMS: [M+H]⁺ 571,569 (³⁷Cl; ³⁵Cl). ¹H NMR (400 MHz, CDCl₃) δ 1.21 (t, 3H), 3.44 (m, 2H),3.79 (s, 3H), 4.73 (s, 2H), 6.45 (s, 1H), 6.65 (t, 1H), 6.80 (d, 2H),7.14 to 7.44 (m, 8H), 6.95 (m 2H).

Step 85-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxy-phenyl)-isoxazole-3-carboxylicacid ethylamide

To an ice-bath cooled solution of5-(2,4-Bis-benzyloxy-5-chlorophenyl)-4-(4-methoxy-phenyl)-isoxazole-3-carboxylicacid ethylamide (0.213 mg, 0.374 mmol) in dichloromethane (5 mL) under anitrogen atmosphere was added a 1.0M solution of Boron trichloride indichloromethane (1.12 mL; 1.12 mmol). The reaction mixture was stirredat 0° C. for 15 minutes then at ambient temperature for 35 minutes. Thereaction mixture was re-cooled to 0° C. and quenched by the addition ofsaturated aqueous sodium hydrogen carbonate solution (5 mL). Afterstirring for 5 minutes the dichloromethane was removed in vacuo and theresidue was partitioned between ethyl acetate (30 mL) and water (30 mL).The phases were separated and the organic phase was washed with water(30 mL), saturated aqueous sodium chloride solution (30 mL) then driedover sodium sulphate. The mixture was filtered and the filtrate solventswere removed in vacuo to afford a foam-like colourless solid which waspurified by adsorption onto silica gel then flash chromatography onsilica gel (10 g IST) eluting with 50% ethyl acetate in hexane. Thisaffords5-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxy-phenyl)-isoxazole-3-carboxylicacid ethylamide as a colourless solid (0.097 g; 67%). LCMS: [M+H]⁺ 391,389 (³⁷Cl; ³⁵Cl). ¹H NMR (400 MHz, d₆-DMSO) □ 1.08 (t, 3H), 3.22 (m,2H), 3.73 (s, 3H), 6.59 (s 1H), 6.87 (d, 1H), 7.13-7.17 (m, 3H), 8.88(br t, 1H), 10.09 (s, 1H), 10.62 (s, 1H).

Example 25 had activity ‘A’ in the Fluorescence Polarisation Assay, asdescribed below.

Similarly, Example 26 was prepared by coupling the Boc protected4-piperazinophenyl boronate ester as above. This boronate ester was madefrom 1-(4-bromophenyl)piperazine by boc protection followed by boronateester formation by Pd-catalysed coupling with bis(tetramethylpinacolato)diboron. Example 27 was made similarly. Example 27a was made bydeprotection of5-(2,4-Bis-benzyloxy-5-chlorophenyl)-4-bromo-isoxazole-3-carboxylic acidethylamide:

Hsp90 Example Structure MH+ IC50* 26 

443 A 27 

377 A 27a

362 A *Fluorescence Polarisation Assay

Scheme 7: Preparation of5-(2,4-bis-benzyloxy-5-chloro-phenyl)-4-iodo-3-methyl-isoxazoleintermediate

Example 284-Chloro-6-[3-methyl-4-(3-morpholin-4-ylmethyl-phenyl)-isoxazol-5-yl]-benzene-1,3-diol

Step 1 1-(2,4-Bis-benzyloxy-5-chloro-phenyl)-3-hydroxy-but-2-en-1-one

To a solution of ketone (15 g) in EtOAc (200 ml) was added sodium metal(3.0 g) in small pieces. The suspension was stirred at room temperaturefor 15 mins, then heated to reflux overnight. The reaction was quenchedwith acetic acid, and the yellow precipitate filtered. This wastriturated in hexanes to give bright yellow crystals. NMR indicated thiswas the required product—mostly in enol form—small trace of keto form.

Step 2 5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-3-methyl-isoxazole

The diketone (4.0 g) was suspended in 80% aq EtOH. Hydroxylaminehydrochloride (3.4 g) and sodium acetate (4.0 g) was added and the pHadjusted to 8/9 with 2M NaOH. The solution was refluxed for 24 hrs(difficult to monitor by TLC due to very similar R_(f) values). Afterthis time the solution was acidified to pH5 with 1M HCl and poured intowater. The white precipitate was filtered, washed with water, andtriturated with hexane to give a white solid. Notes; Compound can alsobe washed with ether if necessary to remove trace impurities but notusually required. NMR indicated this to be the required product.

Step 3 5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-iodo-3-methyl-isoxazole

Isoxazole (2 g) was placed in a mixture of acetic acid (24 ml) and water(30 ml). Iodinemonochloride (2 g excess) was added and the solutionheated at 80° C. for 2-3 hrs. After cooling to room temperature 10%Na₂SO₃ (Sodium sulphite) in water was added (50 ml). A viscous orangesolid/oil was separated from the mixture and was washed with water. Itwas then dissolved in acetone and filtered. Removal of the acetone undervacuum gave a sticky orange oil which solidified to a orange solidovernight. NMR and LCMS indicated this was the required product.

Step 43-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-3-methyl-isoxazol-4-yl]-benzaldehyde

5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-iodo-3-methyl-isoxazole (200 mg,0.38 mmol), and 3-formylbenzene boronic acid (85 mg, 1.5 equiv.) weredissolved in DMF (12 ml) before 1M Sodium hydrogen carbonate solution(1.1 ml, 3.0 equiv) and Pd(Ph₃P)₂Cl₂ (21 mg, 0.08 equiv.) were addedwith stirring. The reaction mixture was transferred to three microwavetubes which were sealed and the mixtures within degassed before beingirradiated by an initial power of 200 W to a temperature of 150° C. for15 minutes in a CEM microwave apparatus. Upon cooling the reactionmixtures were combined and partitioned between ethyl acetate (10 ml) andwater (10 ml). The aqueous layer was separated and extracted again withethyl acetate (10 ml). The organics were then combined washed with water(2×20 ml), brine (20 ml), dried over Na₂SO₄ before being condensed invacuo and purified by flash chromatography on silica gel, eluting with25% ethyl acetate in hexane.

LCMS t_(R)=9.06, MS m/z 510.4 [M+H]⁺

Step 54-{3-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-3-methyl-isoxazol-4-yl]-benzyl}-morpholine

3-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-3-methyl-isoxazol-4-yl]-benzaldehyde(25 mg, 0.05 mmol) and morpholine (0.3 ml) were mixed with DCE (0.5 ml)in an microwave tube. Sodium triacetoxyborohydride (15 mg, 1.4 equiv)was added, the tube sealed, and nitrogen atmosphere introduced. After 1hr more sodium triacetoxyborohydride (15 mg) was added and the reactionleft stirring overnight. TLC analysis showed that the reaction had notgone to completion so a drop of acetic acid was added and the reactionagain left stirring overnight after which the reaction was quenched with1M NaHCO₃ solution (7 ml) and extracted into EtOAc (5 ml). This wasdried over MgSO₄ and the solvent removed in vacuo to provide 13 mg ofthe crude product as an off white powder which was taken over to thedeprotection step.

Step 64-Chloro-6-[3-methyl-4-(3-morpholin-4-ylmethyl-phenyl)-isoxazol-5-yl]-benzene-1,3-diol

4-{3-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-3-methyl-isoxazol-4-yl]-benzyl}-morpholinewas deprotected as previously shown and the crude purified bypreparative TLC eluting with 10% Ethanol in dichloromethane to provide0.6 mg (7% yield) of the product as a white powder.

LCMS t_(R)=5.46, MS m/z 399.3 [M−H]⁻

Example 28 had activity ‘A’ in the Fluorescence Polarisation Assay, asdescribed below.

Example 291-{3-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-3-methyl-isoxazol-4-yl]-benzyl}-piperidine-4-carboxylicacid amide

Prepared in using a similar procedure to4-chloro-6-[3-methyl-4-(3-morpholin-4-ylmethyl-phenyl)-isoxazol-5-yl]-benzene-1,3-diolexcept that isonipecotamide replaced the morpholine and the sodiumtriacetoxyborohydride (3 equiv.) and acetic acid (1 drop) were addedinitially. The reaction was complete after 18 hrs and the crude obtainedafter work up was taken over to the deprotection step.

1-{3-[5-(5-Chloro-2,4-dihydroxy-phenyl)-3-methyl-isoxazol-4-yl]-benzyl}-piperidine-4-carboxylicacid amide

1-{3-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-3-methyl-isoxazol-4-yl]-benzyl}-piperidine-4-carboxylicacid amide was deprotected as previously shown and the crude purified bypreparative TLC eluting with 10% Ethanol in dichloromethane to provide0.7 mg (3% yield) of the product as a white powder.

LCMS t_(R)=5.36, MS m/z 442.3 [M+H]⁺

Example 29 had activity ‘A’ in the Fluorescence Polarisation Assay, asdescribed below.

In a similar way, example 30 was prepared:

Hsp90 Example Structure MH+ IC50 30

359 A* *Fluorescence Polarisation Assay

Example 31

Step 15-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-iodo-isoxazole-3-carboxylic acidethylamide

5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-isoxazole-3-carboxylic acidethylamide (0.90 g, 1.94 mmol), N-Iodosuccinimide (0.44 g, 1 equiv.) andAmmonium cerium (IV) nitrate (0.53 g, 0.5 equiv) were suspended inAcetonitrile (55 ml) before heating to reflux (oil bath 100° C.) whereupon the mixture became homogeneous. After 18 hrs the solution wascooled and the solvent removed in vacuo to give a thick orange oil. Thiswas partitioned between DCM (25 ml) and water (10 ml), the organic layerwas kept and washed with brine (2×25 ml) before drying over Na2SO4. TheDCM was removed in vacuo to provide 0.88 g (77% yield) of the product asa orange/tan coloured powder.

LCMS t_(R)=8.75, MS m/z 589.1 [M+H]⁺

Step 2 1-(3-Bromo-phenyl)-4-methyl-piperazine

1,3-Dibromobenzene (0.90 ml, 7.49 mmol), N-methylpiperazine (0.28 ml,2.50 mmol) and anhydrous toluene (7 ml) were added by syringe to a dry,argon filled flask. The solution was thoroughly mixed before BINAP (47mg) and Pd₂dba₃ (23 mg) were delivered and the flask refilled with Argonand DBU (0.93 g, 2.5 equiv.) added via syringe. The reaction mixture waswarmed to 60° C. before freshly ground sodium tertbutoxide was added inone portion to start the reaction. The reaction was left stirring at 60°C. overnight and the TLC analysis appeared to show that some piperazinewas still present so the reaction was heated to 100° C. and stirred foranother 24 hrs after which it was partitioned between EtOAc (20 ml) andwater (20 ml). The aqueous layer was extracted again with EtOAc and thecombined organics were washed with 1.6M HCl solution (2×10 ml). Theacidic solution containing the product was then basified first with asimilar volume of 1M NaOH solution to acid solution and then carefullysolid sodium bicarbonate was added to make the pH=8.5 before extractionback into EtOAc (2×15 ml), which was washed with brine, dried over MgSO₄and evaporated to dryness to provide 0.50 g (78% yield) of the pureproduct as a yellow oil.

LCMS t_(R)=4.55, MS m/z 255.4/257.3 [M+H]⁺

Step 31-Methyl-4-[3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-piperazine

To a solution of PdCl₂ (dppf).DCM (10 mg, 0.012 mmol) in anhydroustoluene (4 ml) in an argon filled sealed microwave tube was added the1-(3-Bromo-phenyl)-4-methyl-piperazine (100 mg, 0.39 mmol), Et₃N (0.11ml, 2 equiv.), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.09 ml, 1.5equiv). The microwave tube was evacuated and backfilled with Argonbefore being irradiated in a CEM Microwave reactor at 100° C. for 1 hrusing an initial power of 200 W. The reaction mixture was partitionedbetween more toluene (6 ml) and water (10 ml), the organic layerseparated, washed with water (1×10 ml), dried over MgSO₄ and thenevaporated in vacuo to leave a purple/brown residue which was used forsuzuki coupling without further purification.

LCMS t_(R)=0.97, MS m/z 303.5 [M+H]⁺

Step 45-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-[3-(4-methyl-piperazin-1-yl)-phenyl]-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-bromo-isoxazole-3-carboxylicacid ethylamide (38 mg, 0.07 mmol) and1-Methyl-4-[3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-piperazine(31 mg, 2 equiv.) were coupled together using the suzuki methodpreviously described to provide 37 mg (83% yield) of the crude as abrown oil which was taken on to the deprotection step.

Step 55-(5-Chloro-2,4-dihydroxy-phenyl)-4-[3-(4-methyl-piperazin-1-yl)-phenyl]-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-chloro-phenyl-4-[3-(4-methyl-piperazin-1-yl)-phenyl]-isoxazole-3-carboxylicacid ethylamide was deprotected as previously shown. The precipitateformed during the reaction was separated, partitioned between EtOAc andwater. The aqueous layer was kept, basified using solid sodium hydrogencarbonate and the product extracted using EtOAc (2×10 ml). The combinedorganics were washed with brine (10 ml) dried over MgSO₄ and evaporatedin vacuo to provide 5.2 mg (20% yield) of product as a tan colouredpowder.

LCMS t_(R)=5.58, MS m/z 457.3 [M+H]⁺

δ_(H) (d⁴-MeOH), 7.17 (1H, m, Ar—H), 7.09 (1H, s, Ar—H), 6.94 (1H, m,Ar—H), 6.80 (1H, m, Ar—H), 6.49 (1H, s, Ar—H), 3.13 (4H, t,NCH₂CH₂N—CH₃), 2.69 (2H, q, CONHCH₂CH₃), 2.61 (4H, t, NCH₂CH₂N—CH₃),2.37 (3H, s, NCH₂CH₂N—CH₃), 1.19 (3H, t, CONHCH₂CH₃).

Example 31 had activity ‘A’ in the Fluorescence Polarisation Assay, asdescribed below.

Examples 32-38 in the Table below were prepared similarly, but with thefollowing variations:

1. For Example 36, the dioxaborolan intermediate was prepared asfollows:

Step 1 1-(4-Bromo-phenyl)-4-methyl-piperazine

1-(4-Bromo-phenyl)-piperazine (1 g, 4.1 mmol) and potassium carbonate(1.8 g, 3 eq) in DMF (15 ml) treated with methyl iodide (250 μl, 1.1equivalents), solution stirred at room temperature overnight. Reactionquenched with deionised water (10 ml), extracted with ethyl acetate.Organic phase washed with sodium hydrogen carbonate to remove anydimethylated impurity, dried and solvent removed to give1-(4-Bromo-phenyl)-4-methyl-piperazine in 73% yield.

LC retention time 2.21 minutes [M+H]⁺ 256 (Run time 3.75 mins).

Step 21-Methyl-4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-piperazine

1-(4-Bromo-phenyl)-4-methyl-piperazine (750 mg, 3 mmol) in DMSO (15 ml)with bis(pinacolato)diboran (1.1 g, 1.5 equivalents) and potassiumacetate (900 mg, 3 equivalents). Suspension degassed before treatmentwith PdCl2 (dppf) (cat.), stirred at 80 C. Additionalbis(pinacolato)diboran (1 eq) added after 3 hours, stirred for a further2 hours. Suspension partitioned between ethyl acetate and water.Purification by column chromatography 0-8% methanol gradient indichloromethane to give1-Methyl-4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-piperazinein 62% yield.

LC retention time 1.83 minutes [M+H]+ 303 (Run time 3.75 mins).

2. For examples 37 and 38, a boronic acid intermediate was used insteadof a diaoxaborolan, the former being prepared as follows:

4-[(2-Methylsulfonyl)-ethylaminomethyl]-phenyl boronic acid(Intermediate for Example 37)

4-Aminomethyl phenyl boronic acid hydrochloride (560 mg, 3 mmol) inethanol (5 ml) was treated with methyl vinyl sulfone (260 μl, 1equivalent) and triethyl amine (1.2 ml, 3 equivalents). The solution wasstirred at 100° C. for 2 hrs. Ethanol removed under vacuum, partitionedin water and butanol to give4-[(2-methylsulfonyl)-ethylaminomethyl]-phenyl boronic acid in 94%yield.

LC retention time 0.39 minutes [M+H]+ 258 (Run time 3.75 mins).

4-[N-methy S,S-dioxo-thiomorpholino]-phenyl boronic acid (Intermediatefor Example 38)

4-Aminomethyl phenyl boronic acid hydrochloride (456 mg, 2.4 mmol) inethanol (8 ml) treated with vinyl sulfone (244 μl, 1 equivalent) andtriethylamine (2 equivalents), solution stirred at 100 C for 3 hrs.Ethanol removed under vacuum, partitioned in water and butanol to givethe product in 88% yield.

LC retention time 1.65 minutes [M+H]+ 270 (Run time 8 mins).

Hsp90 Example Structure MH+ IC50* 32

409 411 A 33

410 412 A 34

360 362 A 35

409 411 A 36

457 459 A 37

494 496 A 38

506 508 A

Example 39

Step 15-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(3-chloro-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-bromo-isoxazole-3-carboxylicacid ethylamide (60 mg, 0.11 mmol), and 3-chlorobenzene boronic acid (23mg, 1.3 equiv.) were coupled together using the suzuki method previouslydescribed to provide 35 mg (55% yield) of the crude as a brown powderwhich was taken on to the next step.

Step 2[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(3-chloro-phenyl)-isoxazol-3-ylmethyl]-ethyl-amine

To a solution of5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(3-chloro-phenyl)-isoxazole-3-carboxylicacid ethylamide (36 mg, 0.06 mmol) in anhydrous THF under argon wasadded 1M Borane-THF complex (1 ml) and the solution refluxed overnight.After cooling the solution was poured on to a Isolute® SPE Flash SCX-2 5g column which was quickly eluted with methanol (2×20 ml). The desiredproduct was then recovered by eluting with a mixture of 10% ammonia inmethanol (2×10 ml) which was evaporated in vacuo to provide 23 mg (65%yield) of a light yellow powder.

LCMS (LCT) t_(R)=8.18, MS m/z 558.8 [M+H]⁺

Example 39 had activity ‘A’ in the Fluorescence Polarisation Assay, asdescribed below.

Example 40 was similarly prepared:

Hsp90 Example Structure MH+ IC50* 40

375 377 A *Fluorescence Polarisation Assay

Example 41

Step 15-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-iodo-isoxazole-3-carboxylic acidethylamide (prepared as for Example 31) (2 g, 3.4 mmol), 4-formylboronicacid (0.612 g, 4.08 mmol), NaHCO₃ (10.2 ml, 1M aq. solution, 10.2 mmol),PdCl₂(PPh₃)₂ (119 mg, 0.17 mmol) and DMF (50 ml) were combined. Themixture was then degassed by bubbling N₂ through it for 5 minutes beforebeing heated at 80° C. for 1 hour. The mixture was then evaporated invacuo and partitioned between EtOAc (3×50 ml) and water (50 ml). Thecombined, dried (Na₂SO₄) organics were evaporated in vacuo to give acrude oil. This was dissolved in EtOAc and passed through a plug ofSiO₂, washing through with EtOAc. The filtrate was evaporated in vacuoand the resulting oil triturated with Et₂O to afford5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (1.577 g, 82%) as a pale coloured solid, LC/MS: RT=2.908min. 567.3 (MH⁺).

Step 25-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

Acetic acid (0.37 ml, 6.44 mmol) was added dropwise to a mixture of5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (730 mg, 1.29 mmol), morpholine (0.225 ml, 2.58 mmol), 3A powdered molecular sieves (730 mg) and MeOH (21 ml). This was left tostir overnight under N₂. The mixture was then evaporated in vacuo andthe resultant crude partitioned between CH₂Cl₂ (3×40 ml) and sat. NaHCO₃solution (40 ml). The combined, dried (Na₂SO₄) organics were evaporatedin vacuo to give crude5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (810 mg) as a yellow solid, LC/MS: RT=2.365 min. 638.4(MH⁺).

Step 35-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

BCl₃ (1M sol. in CH₂Cl₂, 3.87 ml, 3.87 mmol) was added dropwise to asolution of the crude5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (810 mg, ˜1.29 mmol) in CH₂Cl₂ (30 ml) at 0° C. Thereaction was then allowed to reach RT. Saturated aqueous NaHCO₃ (40 ml)was then added slowly and the resultant mixture concentrated in vacuo.This was then partitioned between EtOAc (3×50 ml) and water (50 ml). Thecombined, dried (Na₂SO₄) organics were evaporated in vacuo. Flashchromatography eluting with CH₂Cl₂— 10% MeOH/1% NH₃/CH₂Cl₂ afforded5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (380 mg, 64% over 2 steps) as a yellow foam, LC/MS:RT=1.751 min. 458.2 (MH⁺).

Example 41 had activity ‘A’ in the Fluorescence Polarisation Assay, asdescribed below.

In the following Table, Examples 42-64 were prepared by methodsanalogous to Example 41, using the appropriate aldehyde or ketone.

Hsp90 Example Structure MH+ IC50* 42

472 474 A 43

458 460 A 44

472 474 A 45

458 A 46

499 501 A 47

471 473 A 48

471 A 49

444 446 A 50

486 488 A 51

500 502 A 52

456 458 A 53

472 474 A 54

442 A 55

452 A** 56

450 A** 57

465 A** 58

479 481 A 59

416 418 A 60

446 448 A 61

471 473 A 62

499 501 A 63

517 519 A 64

476 478 A *Fluorescence Polarisation Assay **prepared from ethylresorcinol starting material

Additional compounds 41a-s were prepared by methods analogous to Example41:

Example Structure MH+ Hsp90 IC50* 41a

468 A 41b

438 A** 41c

442 A 41e

395 A 41f

474 A 41g

476 A 41h

428 A 41i

470 A 41j

472 A 41k

502 A 41m

458 A 41n

389 A*** 41p

471 A 41q

475 A**** 41r

507 A***** 41s

472 A *Fluorescence Polarisation Assay **prepared from ethyl resorcinolstarting material ***prepared by reduction of the aldehyde intermediate****prepared by alkyation of the intermediate phenol *****prepared fromthe naphthyl aldehyde

Example 65

Step 1 1-(2,4-Bis-benzyloxy-phenyl)-ethanone

35 g of 2,4-dihydroxyacetophenone (0.230 mol, 1 eq) were dissolved in500 ml acetonitrile. 79.5 g of potassium carbonate (0.575 mol, 2.5 eq)and 86.6 g benzyl bromide (0.506 mol, 2.2 eq) were added. The mixturewas refluxed for 64 hours, cooled down and acetonitrile removed underreduced pressure. The residue was separated between water and ethylacetate. The residue was mainly mono-benzylated resorcinol.

The crude product (43 g) was then dissolved in 250 ml DMF. Potassiumcarbonate (29 g, 0.210 mol, 1.2 eq) and 25 ml benzyl bromide (0.210 mol,1.2 eq) were added and the mixture was stirred over night. The solventwas removed under reduced pressure and the residue was separated betweenethyl acetate and water. After removal of the solvent, the residue wastriturated with hexane to remove excess benzyl bromide.

LC-MS [M+H]+=333

Yield: 51.2 g (67%)

Step 2 1-(2,4-Bis-benzyloxy-5-bromo-phenyl)-ethanone

51.2 g of 1-(2,4-Bis-benzyloxy-phenyl)-ethanone (0.154 mol, 1 eq) weredissolved in 250 ml DMF. 27.42 g N-bromosuccinimide (0.154 mol, 1 eq) in100 ml DMF were added dropwise. The mixture was stirred at roomtemperature over night. The reaction mixture was poured onto 700 ml ofwater and the precipitate filtered off. The filter cake was rinsed withwater and the colourless solid was recrystallised from 370 mlacetonitrile.

LC-MS [M+H]+=411 & 413

Yield: 58.15 g (92%)

Step 3 4-(2,4-Bis-benzyloxy-5-bromo-phenyl)-2,4-dioxo-butyric acid ethylester

9.75 g sodium (0.424 mol, 3 eq) were dissolved in 500 ml absoluteethanol (1.5 hours). 58 g of1-(2,4-Bis-benzyloxy-5-bromo-phenyl)-ethanone (0.141 mol, 1 eq) and30.98 g diethyl oxalate (0.212 mol, 1.5 eq) were added and the mixturewas refluxed for 2 hours. After cooling down, the mixture was pouredonto 220 ml of 2N aqueous HCl and the product was extracted into 700 mldichloromethane. The solvent was removed under reduced pressure and theyellow residue was triturated with 150 ml diethyl ether.

Yield: 69.24 g (96%)

1H NMR (400 MHz, CDCl3) δ 1.27 (t, 3H), 4.27 (q, 2H), 5.13 (d, 2H), 6.54(s, 1H), 7.37 (m, 10H), 8.17 (s, 1H).

Step 4 5-(2,4-Bis-benzyloxy-5-bromo-phenyl)-isoxazole-3-carboxylic acidethyl ester

69.3 g of 4-(2,4-Bis-benzyloxy-5-bromo-phenyl)-2,4-dioxo-butyric acidethyl ester (0.135 mol, 1 eq) were dissolved in 750 ml ethanol. 14.11 ghydroxylamine hydrochloride (0.203 mol, 1.5 eq) were added. The mixturewas refluxed for 2.5 hours and cooled down. It was then poured onto 1000ml water, the precipitate was filtered off. The filter cake was washedwith 500 ml of water followed by 75 ml diethyl ether and dried.

Yield: 67.62 g (99%)

1H NMR (400 MHz, CDCl3) δ 1.39 (t, 3H), 4.41 (q, 2H), 5.11 (d, 2H), 5.15(d, 2H), 6.58 (s, 1H), 6.99 (s, 1H), 7.35 (m, 10 OH), 8.16 (s, 1H).

Step 5 5-(2,4-Bis-benzyloxy-5-bromo-phenyl)-isoxazole-3-carboxylic acidethylamide

5-(2, 4-bis-benzyloxy-5-bromo-phenyl)-isoxazole-3-carboxylic acid ethylester was suspended in ethanol and ethylamine (2M in methanol, 3 eq),the resulting yellow suspension was heated to reflux (800° C.) undernitrogen, at which point the reagents went into solution. This washeated for 14 hours, then left to cool to ambient temperature. A whiteprecipitate formed, which was filtered off and washed with furtherethanol before being dried in vacuo.

LC-MS retention time 2.868 minutes [M+H]+=507 & 509 (run time 3.75minutes)

Step 6 5-(2,4-Bis-benzyloxy-5-styrylphenyl)-isoxazole-3-carboxylic acidethylamide

To a mixture of trans-2-phenylvinylboronic acid (0.472 g, 3.2 mmol) and5-(2,4-Bis-benzyloxy-5-bromophenyl)-isoxazole-3-carboxylic acidethylamide (1.079 g, 2.13 mmol) was added sodium hydrogen carbonate (536mg, 6.39 mmol) followed by DMF (25 mL) and water (5 mL). The mixture wasdegassed by evacuation and flushing with nitrogen (three times),followed by bubbling nitrogen gas through mixture for five minutes.Dichlorobis(triphenylphosphine)palladium (II) (149 mg, 0.21 mmol) wasadded and reaction mixture was heated under a nitrogen atmosphere at 80°C. for seven hours (reaction mixture becomes dark brown in colour after10 minutes). The reaction mixture was allowed to cool to ambienttemperature and the majority of solvents were removed in vacuo. Theresulting residue was partitioned between ethyl acetate (100 mL) andwater (100 mL) and this mixture was filtered through a pad of celite toremove Palladium residues. The phases were separated and the organicphase was washed with water (2×50 mL), saturated aqueous sodium chloridesolution (100 mL) then dried over sodium sulphate. The mixture wasfiltered and the filtrate solvents were removed in vacuo to afford abrown solid (800 mg). The celite filter cake was washed withdichloromethane then dried over sodium sulphate. The mixture wasfiltered and the filtrate solvents were removed in vacuo to afford abrown solid (541 mg). The combined product batches were purified bytrituration with ethyl acetate-hexane mixture. This affords5-(2,4-Bis-benzyloxy-5-styrylphenyl)-isoxazole-3-carboxylic acidethylamide as a light brown solid (808 mg, 71%). LCMS: [M+H]+ 531. 1HNMR (400 MHz, CDCl3) δ 1.12 (t, 3H), 3.37 (m, 2H), 4.95 (s, 2H), 5.07(s, 2H), 6.46 (s, 1H), 6.70 (brt, 1H). 7.11 (s, 1H), 7.17 (d, 1H), 7.23(d, 1H), 7.32-7.44 (m, 15H), 8.09 (s, 1H).

Step 7 5-(2,4-Bis-benzyloxy-5-phenethylphenyl)-isoxazole-3-carboxylicacid ethylamide

Palladium on charcoal catalyst (10%; 50 mg) was added to a degassedsolution of 5-(2,4-Bis-benzyloxy-5-styrylphenyl)-isoxazole-3-carboxylicacid ethylamide (690 mg, 1.30 mmol) in 1,4-dioxane (50 mL) under anitrogen atmosphere. The reaction mixture was hydrogenated for a totalof 4.75 hrs with further Pd on charcoal catalyst (50 mg) added at 0.75and 2.5 hrs. The reaction mixture was filtered through a pad of celite,which was washed with 1,4-dioxane (20 mL) and dichloromethane (20 mL).The combined filtrate solvents were removed in vacuo to afford acream-coloured solid, which was purified by flash chromatography onsilica gel (20 g, IST) eluting with 10 to 50% ethyl acetate in hexane.This affords5-(2,4-Bis-benzyloxy-5-phenethylphenyl)-isoxazole-3-carboxylic acidethylamide as a pale yellow solid (609 mg, 88%). LCMS: [M+H]+ 533. 1HNMR (400 MHz, CDCl3) δ 81.26 (t, 3H), 2.86-2.96 (m, 4H), 3.49 (m, 2H),5.03 (s, 2H), 5.18 (s, 2H), 6.56 (s, 1H), 6.81 (t, 1H), 7.07 (s, 1H),7.15-7.20 (m, 3H), 7.23-7.28 (m, 2H), 7.31-7.42 (m, 10H), 7.73 (s, 1H).

Step 85-(2,4-bis-benzyloxy-5-phenethylphenyl)-4-bromo-isoxazole-3-carboxylicacid ethylamide

N-Bromosuccinimide (207 mg, 1.16 mmol) was added to a suspension of5-(2,4-Bis-benzyloxy-5-phenethylphenyl)-isoxazole-3-carboxylic acidethylamide (564 mg, 1.06 mmol) in acetonitrile (20 mL). Ceric ammoniumnitrate (290 mg, 0.53 mmol) was added and the reaction mixture washeated to reflux (affording homogeneous orange solution) and stirred for30 minutes. The reaction mixture was allowed to cool to ambienttemperature and acetonitrile was removed in vacuo. The residue waspartitioned between ethyl acetate (50 mL) and water (50 mL) and thephases were separated. The organic phase was washed with saturatedaqueous sodium chloride solution (50 mL) and dried over sodium sulphate.The mixture was filtered and the filtrate solvents were removed in vacuoto afford a yellow oil which was purified by flash chromatography onsilica gel (20 g, IST) eluting with 10-30% ethyl acetate in hexane. Thisaffords5-(2,4-Bis-benzyloxy-5-phenethylphenyl)-4-bromo-isoxazole-3-carboxylicacid ethylamide as yellow oil (326 mg, 53%). LCMS: [M+H]+ 613, 611.

Step 95-(2,4-bis-benzyloxy-5-phenethyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

To a mixture of 4-morpholin-4-ylmethyl-phenyl pinnacol borane (0.215 g,0.71 mmol) and5-(2,4-Bis-benzyloxy-5-phenethylphenyl)-4-bromo-isoxazole-3-carboxylicacid ethylamide (0.347 g, 0.57 mmol) was added sodium hydrogen carbonate(142 mg, 1.69 mmol) followed by DMF (10 mL) and water (2.0 mL). Themixture was degassed by evacuation and flushing with nitrogen (threetimes), followed by bubbling nitrogen gas through mixture for fiveminutes. Dichlorobis(triphenylphosphine)palladium (II) (40 mg, 0.057mmol) was added and reaction mixture was heated under a nitrogenatmosphere at 80° C. for 5 hours (reaction mixture becomes dark brown incolour). Another 20 mg (0.029 mmol) ofdichlorobis(triphenylphosphine)palladium (II) was added and reactionmixture was heated at 80° C. for 15 hours then allowed to cool toambient temperature. The majority of solvents were removed in vacuo andthe residue was partitioned between ethyl acetate (50 mL) and water (50mL). This mixture was filtered through a pad of celite to removePalladium residues and then the phases were separated and the organicphase was washed with water (2×50 mL), saturated aqueous sodium chloridesolution (50 mL) then dried over sodium sulphate. The mixture wasfiltered and the filtrate solvents were removed in vacuo to afford abrown oil. The crude reaction product was purified by flashchromatography on silica gel (20 g, IST) eluting with a solvent gradientof 30 to 70% ethyl acetate in hexane. This affords5-(2,4-bis-benzyloxy-5-phenethyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as yellow oil (0.110 g, 27%). LCMS: [M+H]+ 708.

Step 105-(2,4-dihydroxy-5-phenethyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride

To an ice-bath cooled solution of5-(2,4-bis-benzyloxy-5-phenethyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (0.109 g, 0.15 mmol) in dichloromethane (4 mL) under anitrogen atmosphere was added a 1.0M solution of Boron trichloride indichloromethane (0.45 mL; 0.45 mmol). The reaction mixture was stirredat 0° C. for 20 minutes then at ambient temperature for 3.5 hours. Thereaction mixture was re-cooled to 0° C. and quenched by the addition ofsaturated aqueous sodium hydrogen carbonate solution (5 mL). Afterstirring for 5 minutes the dichloromethane was removed in vacuo and theresidue was partitioned between ethyl acetate (20 mL) and water (20 mL).The phases were separated and the organic phase was washed with water(20 mL), saturated aqueous sodium chloride solution (20 mL) then driedover sodium sulphate. The mixture was filtered and the filtrate solventswere removed in vacuo to afford a light-brown oil which was purified byadsorption onto silica gel then flash chromatography on silica gel (10 gIST) eluting with 0 to 5% methanol in ethyl acetate. This affords acolourless oil which was triturated with 1.0M HCl in diethyl ethersolution (5 mL) to afford5-(2,4-dihydroxy-5-phenethyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide hydrochloride (0.019 g; 24%). LCMS: [M+H]+ 528. 1H NMR(400 MHz, d6-DMSO) □ 1.08 (t, 3H), 2.60 (m, 4H), 2.90-3.30 (m, 6H), 3.67(m, 2H), 3.87 (m, 2H), 4.30 (s, 2H), 6.46 (s, 1H), 6.84 (s, 1H),7.05-7.49 (m, 5H), 7.40-7.68 (m, 4H), 8.90 (brs, 1H), 9.67 (s, 1H), 9.89(s, 1H), 10.75 (brs, 1H).

Example 65 had activity ‘A’ in the Fluorescence Polarisation Assay, asdescribed below.

The examples in the following Table were prepared by methods analogousto Example 64, and had the activities shown in the FluorescencePolarisation Assay, as described below.

Ex- Structure MH+ Hsp90 ample IC50* 66

457 459 A 67

419 A 68

459 A 69

544 A 70

546 A 71

437 A 72

516 A 73

518 A 74

500 A 75

546 A

The additional examples 75a-v in the following table were also preparedby methods analogues to example 65.

Hsp90 Example Structure MH+ IC50 75a

540 A 75b

498 A 75c

542 A 75d

516 A 75e

544 A 75f

518 A 75g

531 A 75h

532 A 75i

526 A 75k

502 A 75m

512 A 75n

545 A 75p

486 A 75q

531 A 75r

504 A 75s

527 A 75t

500 A 75u

501 13 75v

517 A

Example 76

Step 13-(2,4-Bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-5-methyl-isoxazole

Trimethyloxonium boron trifluoride (Aldrich; 70 mg, 0.47 mmol) was addedto a stirred solution of5-(2,4-Bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-3-methyl-isoxazole(Example 3, Step 1) (120 mg, 0.22 mmol) in dichloromethane (3 ml) andstirring was continued for 3 h. The resulting mixture was concentratedin vacuo to leave a white semisolid, which was mixed with hydroxylaminehydrochloride (70 mg, 1.0 mmol), potassium carbonate (120 mg, 0.87 mmol)and methanol (2 ml), and heated at reflux for 18 h. The reaction mixturewas partitioned between water (20 ml) and ethyl acetate (2×10 ml) andthe combined organic phases were dried over anhydrous magnesium sulphateand evaporated in vacuo to leave an colourless oil. The crude productwas purified by column chromatography, silica (10 g), eluting withhexane, followed by diethyl ether/hexane (1:1), to give3-(2,4-Bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-5-methyl-isoxazoleas a white solid (44 mg, 37%)

LC retention time 5.55 minutes [M+H]⁺ 556.0 and 558.0 (Run time 8.00mins)

N.M.R (Chloroform-d) 7.64 (s ArH) 7.356.76 (m 14 ArH) 6.34 (s ArH) 4.90(s 2CH₂) 4.60 (s 2CH₂) 3.79 (s 3CH₃) 2.46 (s 3CH₃)

Step 24-Bromo-6-[4-(4-methoxy-phenyl)-5-methyl-isoxazol-3-yl]-benzene-1,3-diol

Boron trichloride solution (1M in dichloromethane, 1 ml, 1 mmol) wasadded to a solution of3-(2,4-Bis-benzyloxy-5-bromo-phenyl)-4-(4-methoxy-phenyl)-5-methyl-isoxazole(38 mg, 0.068 mmol) in dichloromethane (1 ml), and stirring wascontinued for 1 h. The reaction mixture was partitioned between water(20 ml) and dichloromethane (2×20 ml) and the combined organic phaseswere dried over anhydrous magnesium sulphate and concentrated in vacuoto leave a brown oil. The crude product was purified by columnchromatography, silica (10 g), eluting with hexane, followed byhexane/diethyl ether (3:1 then 1:1), to give4-Bromo-6-[4-(4-methoxy-phenyl)-5-methyl-isoxazol-3-yl]-benzene-1,3-diolas a colourless oil (11 mg, 43%).

LC retention time 2.52 minutes [M+H]⁺ 376.1 and 378.1 (Run time 3.75mins)

N.M.R (DMSO-d₆) 10.40 (s OH) 9.69 (s OH) 7.22 (ArH) 7.10-6.89 (m 4ArH)6.5 (s ArH) 3.7 (s OCH₃) 2.46 (s CH₃)

This compound had activity ‘A’ in the Hsp90 fluorescence polarizationassay.

Example 76A

The following compound is commercially available (Interbioscreen) andhad activity “B) in the fluorescence polarization assay:

Example Structure MH+ 76A

343

The following compounds were made according to Example 76:

Example Structure MH+ Hsp90 IC50 76B

389 A 76C

458 A

Example 77 Preparation of5-(5-tert-Butyl-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

Step 1 1-(5-tert-Butyl-2,4-dihydroxy-phenyl)-ethanone

Sulphuric acid (4 ml, 75 mmol) was added to a suspension of2,4-dihyroxyacetophenone (22.8 g, 150 mmol) in a mixture of2-methyl-2-propanol (35 g, 470 mmol) and trifluoroacetic acid (80 ml),under a nitrogen atmosphere. The resulting suspension was heated, oilbath temperature 75° C., for ˜3 hrs. to give a pale red solution. Theresulting solution was allowed to cool and poured into ice/water (350ml), to give a pale pink precipitate. The solids were removed byfiltration and washed with water (600 ml) and hexane (200 ml) to give apale pink powder. Dried in vacuo (40° C.), to give1-(5-tert-butyl-2,4-dihydroxy-phenyl)-ethanone as a pale orange powder(28.8 g, 92%).

LC retention time 2.74 minutes [M+H]⁺ 209.1 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.35 (s ArH) 6.05 (s ArH) 7.35 (m 2ArH) 2.35 (s3CH₃) 1.15 (s 9 CH₃)

Step 2 1-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-ethanone

Benzyl bromide (10 ml, 84 mmol) was added to a solution of theacetophenone (13.5 g, 65 mmol) in DMF (50 ml), potassium carbonate (20g, 145 mmol) was added and the suspension stirred, at room temperature,for ˜4 hrs. The resulting suspension was poured into water (200 ml) togive a pale orange precipitate. The solids were removed by filtrationand washed with water. The solids were taken up in dichloromethane (150ml) and the solution was washed with water (2×100 ml) and saturatedaqueous sodium chloride solution (100 ml). The solution was dried overanhydrous sodium sulphate and concentrated to a pale red oil.

The oil was taken up in 2-methyl-2-propanol (100 ml) and potassiumtert-butoxide (7.5 g, 67 mmol) added, to give a pale yellow precipitate,benzyl bromide (8 ml, 67 mmol) was added and the mixture heated underreflux for ˜1 hr. The resulting suspension was allowed to cool andpoured into water (250 ml), to give a pale orange precipitate. Thesolids were removed by filtration and washed with water. The solids weretaken up in ethyl acetate (150 ml) and washed with water (2×200 ml) andsaturated aqueous sodium chloride solution (100 ml). The solution wasdried over anhydrous sodium sulphate and concentrated to a orangesemi-solid, trituration with methanol gave a pale pink solid. Solidswere removed by filtration and dried in vacuo (40° C.), to give1-(2,4-bis-benzyloxy-5-tert-butyl-phenyl)-ethanone as a pale pink powder(9.1 g, 36%).

LC retention time 3.03 minutes [M+H]⁺ 389.3 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.65 (s ArH) 7.25-7.15 (m 10ArH) 6.35 (s ArH) 4.95(s 2CH₂) 4.9 (s 2 CH₂) 2.4 (s 3CH₃) 1.2 (s 9 CH₃)

Step 34-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-2-hydroxy-4-oxo-but-2-enoicacid ethyl ester

Sodium ethoxide (2.8 g, 41 mmol) was added to a suspension of the1-(2,4-bis-benzyloxy-5-tert-butyl-phenyl)-ethanone (7.8 g, 20 mmol) inethanol (40 ml). Diethyl oxalate (4 ml, 29.5 mmol) was added and theresulting suspension heated under reflux for ˜2 hrs. to give a pale redsolution. The solution was allowed to cool and poured into water (200ml), the mixture was acidified with hydrochloric acid (50 ml, 1M) andextracted with dichloromethane (150 ml). The extracts were washed withwater (2×200 ml) and saturated aqueous sodium chloride solution (100ml). The solution was dried over anhydrous sodium sulphate andconcentrated to a yellow gum. Trituration with hexane gave a yellowsolid. Solids were removed by filtration and washed with hexane anddried in vacuo (40° C.), to give4-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-2-hydroxy-4-oxo-but-2-enoicacid ethyl ester as a yellow powder (9.1 g, 93%).

N.M.R (Chloroform-d) 8.0 (s ArH) 7.5-7.35 (m 11ArH) 6.6 (s ArH) 5.2 (s2CH₂) 5.15 (s 2 CH₂) 4.3 (q J 7.1 Hz 2 CH₂) 1.4 (s 9 CH₃) 1.25 (t J 7.1Hz 3CH₃)

Step 4 5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-isoxazole-3-carboxylicacid ethyl ester

Hydroxylamine hydrochloride (3.6 g, 52 mmol) was added to a solution of4-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-2-hydroxy-4-oxo-but-2-enoicacid ethyl ester (9.0 g, 18.5 mmol) in ethanol (75 ml) and thesuspension heated under reflux for ˜4 hrs. The resulting solution wasallowed to cool and poured into water (200 ml) to give an off-whiteprecipitate. The solids were removed by filtration and taken up indichloromethane (150 ml). The solution was washed with water (150 ml)and saturated aqueous sodium chloride solution (50 ml).

The solution was dried over anhydrous sodium sulphate and concentratedto an off-white solid. Solids were washed with hexane and dried in vacuo(40° C.), to give5-(2,4-bis-benzyloxy-5-tert-butyl-phenyl)-isoxazole-3-carboxylic acidethyl ester as a pale brown powder (8.0 g, 89%).

LC retention time 3.13 minutes [M+H]⁺ 486.5 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.85 (s ArH) 7.4-7.25 (m 10ArH) 6.9 (s ArH) 6.5 (sArH) 5.1 (s 2CH₂) 5.0 (s 2 CH₂) 4.35 (q J 7.1 Hz 2 CH₂) 1.4 (s 9 CH₃)1.35 (t J 7.1 Hz 3CH₃)

Step 5 5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-isoxazole-3-carboxylic acidethyl ester (10.0 g, 20.6 mmol) was added to a solution of ethylamine inmethanol (60 ml, 2.0M) and the suspension heated, oil bath temperature75° C., for ˜2 hrs. The resulting solution was allowed to cool andconcentrated to a pale brown oil, dichloromethane (150 ml) was added andthe solution washed with water (100 ml) and saturated aqueous sodiumchloride solution (75 ml). The solution was dried over anhydrous sodiumsulphate and concentrated to a brown oil, solidified on standing (9.9 g,˜quant).

LC retention time 3.02 minutes [M+H]⁺ 485.3 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.8 (s ArH) 7.4-7.2 (m 10ArH) 7.0 (s ArH) 6.75 (brt J 5.4 Hz NH) 6.5 (s ArH) 5.1 (s 2CH₂) 5.0 (s 2 CH₂) 3.4 (dq J 5.4 Hz,7.1 Hz 2 CH₂) 1.35 (s 9 CH₃) 1.15 (t J 7.1 Hz 3CH₃)

Step 65-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide

N-iodosuccinimide (9.09 g, 40 mmol) was added to a suspension of5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-isoxazole-3-carboxylic acidethylamide (9.9 g, 20.4 mmol) in acetonitrile (60 ml). Ammonium ceriumnitrate (0.25 g, 0.46 mmol) was added and the suspension stirred for ˜18hrs. The resulting suspension was concentrated and the residue taken upin dichloromethane (125 ml). The resulting solution was washed aqueoussodium metabisulphite solution (2×100 ml, 5%), water (100 ml) andsaturated aqueous sodium chloride solution (100 ml). The solution wasdried over anhydrous sodium sulphate and concentrated to a pale red gum.Trituration with ethanol (25 ml) gave an off-white solid, solids wereremoved by filtration and washed with ethanol. Dried in vacuo (40° C.),to give5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide as an off-white powder (7.75 g, 62%).

LC retention time 3.07 minutes [M+H]⁺ 611.2 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.45-7.25 (m 11ArH) 6.8 (br t J 5.4 Hz NH) 6.6 (sArH) 5.05 (s 4CH₂) 3.5 (dq J 5.4 Hz, 7.1 Hz 2 CH₂) 1.35 (s 9 CH₃) 1.2 (tJ 7.1 Hz 3CH₃)

Step 75-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

Aqueous potassium phosphate (25 ml, 1.2M) solution was added to asolution of5-(2,4-bis-benzyloxy-5-tert-butyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide (6.1 g, 10 mmol) and 4-formylphenyl boronic acid (2.35g, 15.7 mmol) in 1,4-Dioxan (75 ml), under a nitrogen atmosphere.Dichloro-bis(tri-o-tolyl phosphine)palladium(II) (cat.) was added andthe mixture heated, oil bath temperature 100° C. for ˜1 hr. The mixturewas allowed to cool, and the aqueous layer separated and extracted withethyl acetate (100 ml). The combined organics were concentrated to givea pale brown gum.

The crude product was purified by column chromatography, silica (600ml), eluting with ethyl acetate/hexane (1:3), to give5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as a pale yellow foam (5.18 g, 88%).

LC retention time 3.01 minutes [M+H]⁺ 589.4 (Run time 3.75 mins)

N.M.R (Chloroform-d) 9.75 (s CHO) 7.5 (d J 6.9 Hz 2 ArH) 7.2 (d J 6.9 Hz2 ArH) 7.15-7.0 (m 8ArH) 6.8 (m 2 ArH) 6.65 (br t J 5.4 Hz NH) 6.2 (sArH) 4.8 (s 2CH₂) 4.5 (s 2 CH₂) 3.2 (dq J 5.4 Hz, 7.1 Hz 2 CH₂) 1.1 (s 9CH₃) 1.05 (t J 7. Hz 3CH₃)

Step 85-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

Sodium cyanoborohydride (65 mg, 1.03 mmol) was added to a solution of5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (125 mg, 0.21 mmol), morpholine (50 μl, 0.57 mmol) andacetic acid (cat.) in methanol (4 ml) and the solution stirred for ˜72hrs. Dichloromethane (50 ml) was added and the solution washed withwater (2×50 ml) and saturated aqueous sodium chloride solution (50 ml).The solution was dried over anhydrous sodium sulphate and concentratedto a colourless gum.

The crude product was purified by column chromatography, silica (20 g),eluting with ethyl acetate/hexane (1:1), to give5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as a colourless oil (35 mg, 25%).

LC retention time 2.56 minutes [M+H]⁺ 660.8 (Run time 3.75 mins)

N.M.R (Chloroform-d) 7.35-7.05 (m 15ArH) 6.7 (br t J 5.4 Hz NH) 6.4 (sArH) 4.9 (s 2CH₂) 4.75 (s 2 CH₂) 3.6 (t J 4.5 Hz 4 CH₂) 3 (s 2 CH₂) 3.35(dq J 5.4 Hz, 7.1 Hz 2 CH₂) 2.35 (br s 4 CH₂) 1.15 (t J 7.1 Hz 3CH₃) 1.1(s 9 CH₃)

Step 95-(5-tert-Butyl-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

Boron trichloride (1 ml, 1.0M in dichloromethane) solution was added toa solution of5-(2,4-Bis-benzyloxy-5-tert-butyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (35 mg, 0.05 mmol) in dichloromethane (1 ml) at −20° C.(ice/methanol), under a nitrogen atmosphere. The resulting solution wasstirred at 0° C. (ice/water) for ˜90 mins. Methanol (2 ml) was added andthe solution concentrated to a brown gum.

The crude product was purified by preparative HPLC, to give5-(5-tert-Butyl-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as a white powder (formate salt) (21 mg, 75%).

LC retention time 1.97 minutes [M+H]⁺ 480.5 (Run time 3.75 mins)

N.M.R (DMSO-d₆) 8.8 (t J 5.6 Hz NH) 7.25 (d J 7.2 Hz 2ArH) 7.15 (d J 7.2Hz 2ArH) 6.7 (s ArH) 6.45 (s ArH) 3.45 (br s 4 CH₂) 3.2 (dq J 5.6 Hz,7.2 Hz 2 CH₂) 2.3 (br s 4 CH₂) 1.1 (s 9 CH₃) 1.05 (t J 7.2 Hz 3CH₃)

This compound had activity ‘A’ in the Hsp90 fluorescence polarizationassay.

In a similar manner to the preparation of the compound of example 77,examples 77a-f were prepared.

Hsp90 Example Structure MH+ IC50 77a

480 A 77b

466 A 77c

478 A 77d

493 A 77e

399 A 77f

411 A

Example 78 Preparation of5-(2,4-Dihydroxy-5-isobutyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

Step 1 1-(2,4-Bis-benzyloxy-phenyl)-ethanone

Potassium carbonate (2.5 eq) was added to a solution of2′,4′-dihydroxyacetophenone (1 eq) in acetonitrile (400 mL), and thesuspension stirred at room temperature. Benzyl bromide (2.5 eq) wasadded drop wise over 10 minutes and the mixture heated at reflux for 18hours. The mixture was cooled and evaporated in vacuo to give slurry.The slurry was partitioned between water and ethyl acetate, and thelayers were separated. The aqueous layer was further extracted withdichloromethane and the organic extracts were combined, dried (MgSO₄)and evaporated in vacuo. The product was triturated with hexane,filtered and washed with cold hexane and dried in vacuo at 45° C. togive 1-(2,4-Bis-benzyloxy-phenyl)-ethanone as a white powder.

LC retention time 2.704 min [M+H]⁺ 333.3

Step 2 2,4-Bis-benzyloxy-1-isopropenyl-benzene

Methyltriphenylphosphonium bromide (1.1 eq) was suspended in an. THF andcooled to 0° C. under nitrogen. 1.6M ^(n)Butyllithium in hexanes (1.1eq) was added drop wise, and stirred for 30 minutes.1-(2,4-Bis-benzyloxy-phenyl)-ethanone (1 eq) was dissolved in an. THFand added drop wise to the suspension. When addition was completed, theice bath was removed and the reaction mixture was stirred at roomtemperature under nitrogen overnight. Methanol was added to the reactionmixture and the resulting solution was evaporated in vacuo. Hexane wasadded to the resulting oil and heated to reflux for 30 minutes, thenfiltered through Celite. The liquor was evaporated in vacuo to give anoil which was purified by column chromatography, eluting with 30% EtOAcin hexane, to give 2,4-Bis-benzyloxy-1-isopropenyl-benzene.

R_(f) retention time 0.722, 3:1 Hexane: EtOAc.

Step 3 4-Isopropyl-benzene-1,3-diol

2,4-Bis-benzyloxy-1-isopropenyl-benzene was taken up in solution inethanol and added to 10% palladium on carbon, which had been pre-wettedwith water. Hydrogen was introduced to the flask and the mixture wasallowed to shake for 16 hours. The catalyst was filtered from thereaction mixture, by a suitable method, and the liquor was concentratedin vacuo, to give 4-isopropyl-benzene-1,3-diol as a white crystallinesolid.

LC retention time 2.088 min [M+H]⁺ 153.1

Step 4 1-(2,4-Dihydroxy-5-isopropyl-phenyl)-ethanone

4-Isopropyl-benzene-1,3-diol (1 eq) was taken up in BF₃.OEt₂ (6 eq) andacetic acid was added (2 eq). The solution was heated for 16 hours at90° C. than allowed to cool to room temperature. The solution was addeddrop wise to 10% NaOAc (aq) and allowed to stand for 4 hours, beforebeing extracted in to EtOAc. The organic phases were combined and washedwith sat. NaHCO₃ (aq), then dried over MgSO₄, filtered and concentratedin vacuo. The residual oil was purified by column chromatography,eluting with dichloromethane, to give1-(2,4-Dihydroxy-5-isopropyl-phenyl)-ethanone as a white solid.

LC retention time 2.633 min [M+H]⁺ 195.1

Step 5 1-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-ethanone

1-(2,4-Dihydroxy-5-isopropyl-phenyl)-ethanone (1 eq) was dissolved inDMF and potassium carbonate (2.2 eq) then benzyl bromide (2.2 eq) wereadded. The suspension was heated, with stirring to 150° C., undernitrogen, for 16 hrs. The solution was cooled to room temperature andthe mixture was poured into 1 MHCl (aq) then extracted in to ethylacetate. The organic phases were combined and washed again with 1 MHCl(aq) then five times with brine solution. The organic phase was driedover MgSO4, filtered and concentrated in vacuo, to give a solid, whichwas purified by diethyl ether hexane (1:1) trituration to give1-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-ethanone.

LC retention time 3.575 min [M+H]⁺ 375.2

Step 64-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-2-hydroxy-4-oxo-but-2-enoicacid ethyl ester

Sodium (2.8 eq) was added to ethanol under nitrogen at room temperatureand stirred for 25 minutes to generate sodium ethoxide.1-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-ethanone (1 eq) was dissolvedin further ethanol and added to the sodium ethoxide solution. Diethyloxalate (1.64 eq) was added and the reaction mixture heated to refluxfor 4 hours. The mixture was allowed to cool to room temperature andenough 1 MHCl (aq) was added to acidify the reaction mixture, which wasthen concentrated in vacuo. The resulting gum was partitioned betweendichloromethane and brine, and the organic phase was dried over MgSO₄,filtered and evaporated in vacuo to give4-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-2-hydroxy-4-oxo-but-2-enoicacid ethyl ester as a yellow gum.

LC retention time 3.057 min [M+H]⁺ 475

Step 7 5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-isoxazole-3-carboxylicacid ethyl ester

4-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-2-hydroxy-4-oxo-but-2-enoicacid ethyl ester (1 eq) was dissolved in ethanol with stirring.Hydroxylamine hydrochloride (1.2 eq) was added and the solution washeated to reflux for 4 hours under a nitrogen atmosphere. The reactionmixture was cooled to room temperature and concentrated in vacuo. Theresidue was partitioned between brine and dichloromethane. The organicphase was dried over MgSO₄, filtered and concentrated in vacuo to give5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-isoxazole-3-carboxylic acidethyl ester as a solid.

LC retention time 3.059 min [M+H]⁺ 472

Step 8 5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-isoxazole-3-carboxylic acidethyl ester was dissolved in excess 2M ethylamine in methanol and heatedin the Smith Synthesiser microwave at 120° C. for 600 seconds. Thesolution was concentrated in vacuo to give a solid which was purified byhexane trituration, to give5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-isoxazole-3-carboxylic acidethylamide.

LC retention time 2.979 min [M+H]⁺ 471.3

Step 95-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-isoxazole-3-carboxylic acidethylamide (1 eq) was dissolved in an. acetonitrile andN-iodosuccinimide (2.0 eq), followed by ceric ammonium nitrate (0.05 eq)were added, and the solution was stirred at room temperature overnight.The reaction mixture was concentrated in vacuo and the resulting gum waspartitioned between ethyl acetate and brine. The organic phase was driedover MgSO₄, filtered and concentrated in vacuo. The residue was purifiedby column chromatography, eluting with 9:1 hexane: ethyl acetate, togive5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide as an oil.

LC retention time 2.975 min [M+H]⁺ 597.2

Step 105-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide (1 eq) was dissolved in an. DMF. 1 MNa₂CO₃ (aq) wasadded, followed by 4-formylphenylboronic acid (2 eq) and then catalyticPdCl2 (PPh3)2. Nitrogen was bubbled through the solution for ten minutesat ambient temperature, after which time, the temperature was elevatedto 80° C. under a nitrogen atmosphere, for 15 minutes. The reactionmixture was allowed to cool to room temperature and the reaction mixturewas diluted with ethyl acetate. This solution was washed with brine,then dried over MgSO₄, filtered and concentrated in vacuo to give anoil. Purified by column chromatography, eluting with 10% EtOAc inhexane, to give5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide as a white solid.

LC retention time 2.981 min [M+H]⁺ 575.3

Step 115-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide (1 eq) was dissolved in methanol and powdered 3 A sieveswere added. Morpholine (2 eq) was added, followed by sodiumcyanoborohydride (2 eq). Acetic acid (5 eq) was added drop wise and thesuspension was stirred under nitrogen at ambient temperature for 16hours. The reaction mixture was diluted with DCM and washed with sat.NaHCO₃(aq). The organic phase was dried over MgSO₄, filtered andconcentrated in vacuo. The resulting gum was purified by flashchromatography, eluting with 1% MeOH in DCM to give5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as a colourless oil.

LC retention time 4.42 min [M+H]⁺ 646.2 method B

Step 125-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (1 eq) was dissolved in an. DCM and under a nitrogenatmosphere, was cooled to 0° C. 1 MBCl₃ in DCM was added drop wise andthe solution was stirred under these conditions for 30 minutes. Methanol(2 ml) was added and the reaction mixture was concentrated in vacuo.Purification of the sample by preparative LC/MS gave5-(2,4-dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as a white solid.

LC retention time 1.991 min [M+H]⁺ 466.3

This compound had activity ‘A’ in the Hsp90 fluorescence polarizationassay.

In a similar manner to the preparation of the compound of example 78,examples 78a-u were prepared.

Hsp90 Example Structure MH+ IC50 78a

464 A 78b

452 A 78c

479 A 78d

424 A 78e

439 A 78f

680 A 78g

636 Pro-drug see Example 78v 78h

550 Pro-drug see Example 78v 78i

478 A 78j

464 A 78k

480 A 78l

452 A 78m

454 A 78n

495 A 78p

465 A 78q

479 A 78r

608 Pro-drug see Example 78v 78s

480 A 78t

493 A 78u

466 A 78w

492 A 78y

478 A 78z

466 A 78aa

514 A 78ab

478 A 78ac

500 A 78ad

495 A 78ae

521 A 78af

479 A 78ag

481 A 78ah

481 A 78ai

608 Pro- drug see Example 78v

Example 78v Phosphoric acid4-chloro-5-(diethoxy-phosphoryloxy)-2-[3-ethylcarbamoyl-4-(4-methoxy-phenyl)-isoxazol-5-yl]-phenylester diethyl ester

To a solid mixture of5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-methoxy-phenyl)-isoxazole-3-carboxylicacid ethylamide (11 mg, 2.1×10⁻² mmol) and MgO (25 mg) in a small vial,10 drops of diethyl chlorophosphate was added. The resulting mixture washeated and stirred at 70° C. for an hour, the progress of the reactionwas monitored by TLC. When cooled, MeOH (1 ml) and DCM (1 ml) wereadded. After filtration, the solvents were evaporated and yellow oil wasobtained. The di-phosphoryl ester was separated by preparative TLC,yielding 4 mg. R_(f)=0.35; ¹H NMR δ=7.95 (1H, s, broad); 7.74 (1H, s);7.55 (1H, s); 7.32 (2H, d, J=9.0 Hz); 6.90 (2H, d, J=9.0 Hz); 4.30 (8H,q); 3.80 (3H, s); 3.40 (2H, q); 1.35 (12H, t) and 1.25 (3H, t). LCMS:(M+1)⁺=661.1 (RT=7.60 min.)

Example 79 Preparation of5-(2,4-Dihydroxy-5-isobutyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

Step 1 1-(2,4-Dihydroxy-phenyl)-2-methyl-propan-1-one

Resorcinol (1 eq) was taken up in BF₃.OEt₂ (6 eq) and isobutyric acid (1eq) added. The solution was heated for 1.5 hours at 90° C. than allowedto cool to room temperature. The solution was added drop wise to 10%NaOAc (aq) and allowed to stand for 4 hours, before being extracted into EtOAc. The organic phases were combined and washed with sat. NaHCO₃(aq), then dried over magnesium sulfate, filtered and concentrated invacuo to give 1-(2,4-dihydroxy-phenyl)-2-methyl-propan-1-one as a redoil which was used without additional purification

LC retention time 2.279 min [M+H]⁺ 181.1

Step 2 4-Isobutyl-benzene-1,3-diol

Ethyl chloroformate (3 eq) was added slowly to a cooled (0° C.) solutionof 1-(2,4-dihydroxy-phenyl)-2-methyl-propan-1-one (1 eq) andtriethylamine (3 eq) in THF. The mixture was warmed to ambienttemperature and stirred for three hours before being filtered and thesolids washed with cold THF. The combined filtrates were cooled to 0° C.and sodium borohydride (4 eq) in a volume of water equal to the THFfiltrates added slowly. The mixture was warmed to ambient temperature,stirred for three hours and diluted with water. The mixture was twiceextracted with diethyl ether, the combined extracts concentrated todryness and re-suspended in 10% aqueous sodium hydroxide solution (4eq). After refluxing for 90 minutes, the mixture was cooled, acidifiedwith 5M aq HCl and twice extracted with diethyl ether. The organicextracts were dried over magnesium sulphate, filtered and concentratedto dryness to give 4-isobutyl-benzene-1,3-diol as a cloudy oil, whichwas used without further purification.

NMR consistent with structure.

Example 3 1-(2,4-Dihydroxy-5-isobutyl-phenyl)-ethanone

4-Isobutyl-benzene-1,3-diol (1 eq) was taken up in BF₃.OEt₂ (6 eq) andacetic acid (2 eq) was added. The solution was heated for 16 hours at90° C. than allowed to cool to room temperature. The solution was addeddrop wise to 10% NaOAc (aq) and allowed to stand for 4 hours, beforebeing extracted twice with diethyl ether. The organic phases werecombined and washed with sat. NaHCO₃ (aq), then dried over magnesiumsulfate, filtered and concentrated in vacuo to give1-(2,4-dihydroxy-5-isobutyl-phenyl)-ethanone, which was used withoutadditional purification.

NMR consistent with structure.

Step 4 1-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-ethanone

1-(2,4-Dihydroxy-5-isobutyl-phenyl)-ethanone (1 eq) was dissolved in DMFand potassium carbonate (4.4 eq) then benzyl bromide (4.4 eq) was added.The suspension was heated, with stirring to 150° C., under nitrogen, for16 hrs. The solution was cooled to room temperature, filtered andconcentrated to dryness. This solid was purified column chromatography(silica, hexanes:ethyl aceate 4:1) then re-crystallised from ethylacetate:hexanes to give 1-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-ethanoneas colourless crystals.

LC retention time 3.030 min [M+H]⁺ 389.3

Step 5 4-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-2,4-dioxo-butyric acidethyl ester

Sodium (3 eq) was added to ethanol under nitrogen at room temperatureand stirred until complete dissolution occurred.1-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-ethanone (1 eq) was added,followed by diethyl oxalate (1.5 eq) and the reaction mixture heated toreflux for 4 hours. The mixture was allowed to cool to room temperatureand acidified with 2M HCl (aq) to give a yellow precipitate of4-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-2,4-dioxo-butyric acid ethylester, which was obtained by filtration.

LC retention time 3.254 min [M+H]⁺ 489.3

Step 6 5-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-isoxazole-3-carboxylicacid ethyl ester

4-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-2,4-dioxo-butyric acid ethylester (1 eq) was dissolved in ethanol with stirring. Hydroxylaminehydrochloride (1.2 eq) was added and the solution was heated to refluxfor 2 hours. The reaction mixture was cooled to room temperature, togive a precipitate. This precipitate was obtained by filtration to give5-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-isoxazole-3-carboxylic acidethyl ester as a white solid.

LC retention time 3.261 min [M+H]⁺ 486.3

Step 7 5-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-isoxazole-3-carboxylic acidethyl ester was dissolved in 2M ethylamine in methanol (10 eq) andheated in the Smith Synthesiser microwave at 120° C. for 600 seconds.The solution was concentrated in vacuo to give5-(2,4-bis-benzyloxy-5-isobutyl-phenyl)-isoxazole-3-carboxylic acidethylamide as a white solid which was used without additionalpurification.

LC retention time 3.112 min [M+H]⁺ 485.3

Step 85-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-isoxazole-3-carboxylic acidethylamide (1 eq) and N-iodosuccinimide (2.0 eq), were dissolved inacetonitrile, ceric ammonium nitrate (0.1 eq) added and the solution wasstirred at room temperature overnight. The reaction mixture wasconcentrated in vacuo and the resulting gum was partitioned betweenethyl acetate and brine. The organic phase was dried over magnesiumsulfate, filtered and concentrated in vacuo. The residue was purified bycolumn chromatography, eluting with 4:1 hexane: ethyl acetate, to give5-(2,4-bis-benzyloxy-5-isobutyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide as an oil.

LC retention time 3.089 min [M+H]⁺ 611.2

Step 95-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-4-iodo-isoxazole-3-carboxylicacid ethylamide (1 eq) was dissolved in DMF and 1M Na₂CO₃ (aq) (3 eq)was added, followed by 4-formylphenylboronic acid (2 eq) and catalyticPdCl₂(PPh₃)₂. Nitrogen was bubbled through the solution for ten minutesat ambient temperature, after which time, the temperature was elevatedto 80° C. under a nitrogen atmosphere, for 2 hours. The reaction mixturewas allowed to cool to room temperature and the reaction mixture wasdiluted with ethyl acetate. This solution was washed with brine, thendried over magnesium sulfate, filtered and concentrated in vacuo to givean oil which was purified by column chromatography, eluting with 10%EtOAc in hexane, to give5-(2,4-bis-benzyloxy-5-isobutyl-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as a white solid.

LC retention time 5.57 min [M+H]⁺ 589.1 method B

Step 105-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-4-(4-formyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (1 eq) was dissolved in methanol and powdered 3 Å sieveswere added. Morpholine (2 eq) was added, followed by acetic acid (5 eq).After stirring for 30 minutes, sodium cyanoborohydride (2 eq) was addedportionwise and the suspension was stirred under nitrogen at ambienttemperature for 16 hours. The reaction mixture was filtered throughcelite and concentrated to dryness. Column chromatography, eluting with5% MeOH in DCM gave5-(2,4-bis-benzyloxy-5-isobutyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as a colourless oil.

LC retention time 4.53 min [M+H]⁺ 660.2 method B

Step 115-(2,4-Dihydroxy-5-isobutyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide

5-(2,4-Bis-benzyloxy-5-isobutyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (1 eq) was dissolved in an. DCM and under a nitrogenatmosphere, was cooled to 0° C. 1M BCl₃ in DCM (9 eq) was added dropwiseand the solution was stirred for 30 minutes. Methanol (2 ml) was addedand the reaction mixture was concentrated in vacuo. Purification of thesample by preparative LC/MS gave5-(2,4-dihydroxy-5-isobutyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide as a white solid.

LC retention time 1.902 min [M+H]⁺ 480.3

This compound had activity ‘A’ in the Hsp90 fluorescence polarizationassay.

In a similar manner to the preparation of the compound of example 79,example 80 was prepared. Purification of the sample by preparative LC/MSgave the compound as a white solid

Ex- Hsp90 ample Structure MH+ IC50 80

480 A

Example 81N-[5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-fluoro-phenyl)-isoxazol-3-ylmethyl]-methanesulfonamide

Example 82 N-[5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-fluoro-phenyl)-isoxazol-3-ylmethyl]-acetamide5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-fluoro-phenyl)-isoxazole-3-carboxylicacid amide

5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-iodo-isoxazole-3-carboxylic acidamide (0.45 g, 0.80 mmol) was cross coupled to 4-fluorophenylboronicacid (0.17 g, 1.5 equiv.) using the standard conditions described above.The crude product, an orange solid (0.40 g), was taken on to the nextstep without further purification.

LCMS (LCQ) t_(R)=8.70, MS m/z 529.1 [M+H]

C-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-fluoro-phenyl)-isoxazol-3-yl]-methylamine

To a solution of5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-fluoro-phenyl)-isoxazole-3-carboxylicacid amide (0.40 g, 0.76 mmol) in anhydrous THF (20 ml) under argon wasadded 1M Borane-THF complex (1 ml) and the solution refluxed overnight.After cooling the reaction was quenched with methanol (10 ml) and theproduct purified using a Isolute® SPE Flash SCX-2 5 g to provide 0.30 g(77% yield) as a powder.

LCMS (LCQ) t_(R)=7.54, MS m/z 515.2 [M+H]⁺

N-[5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-fluoro-phenyl)-isoxazol-3-ylmethyl]-methanesulfonamide

C-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-fluoro-phenyl)-isoxazol-3-yl]-methylamine(100 mg, 0.19 mmol) was dissolved in DCM (3 ml) before the addition ofmethane sulfonyl chloride (17 μl, 1.1 equiv.) and triethylamine (30 μl,1.1 equiv.). The solution was stirred at room temperature overnightbefore evaporated to dryness in vacuo leaving a the crude benzylprotected product as a blue coloured residue (90 mg). This wasdeprotected using the standard procedure with boron trichloridedescribed above and purified by preparative TLC (10% ethanol in DCM) andsoxhlet extraction of the silica by ether gave the pure compound as anear colourless solid (8 mg, 10% yield).

LCMS (LCQ) tR=6.65, MS m/z 411.2 [M−H]−

δH (d4-MeOH), 7.19 (2H, m, Ar—H), 7.04 (1H, s, Ar—H), 7.03 (2H, m,Ar—H), 6.34 (1H, s, Ar—H), 4.27 (2H, s, CH2NH), 2.81 (3H, s, SO2CH3).

N-[5-(5-Chloro-2,4-dihydroxy-phenyl)-4-(4-fluoro-phenyl)-isoxazol-3-ylmethyl]-acetamide

To a solution ofC-[5-(2,4-Bis-benzyloxy-5-chloro-phenyl)-4-(4-fluoro-phenyl)-isoxazol-3-yl]-methylamine(100 mg, 0.19 mmol) in DCM was added acetic anhydride (130 μl, 7.0equiv.) and triethylamine (81 μl, 3.0 equiv.). The solution was stirredat room temperature until the amine was consumed. The solvent wasremoved in vacuo to leave the yellow tinged oily crude benzyl protectedproduct. This was deprotected using the standard procedure with borontrichloride described above and purified by preparative TLC and soxhletextraction of the silica by ether gave the pure compound as a colourlesssolid (10 mg, 14% yield).

LCMS (LCQ) t_(R)=6.57, MS m/z 377.1 [M+H]⁺

δ_(H) (d⁴-MeOH), 7.17 (2H, m, Ar—H), 7.01 (1H, s, Ar—H), 6.98 (2H, m,Ar—H), 6.32 (1H, s, Ar—H), 4.37 (2H, s, CH₂NH), 1.77 (3H, s, COCH₃).

Examples 83, 84 and 855-(5-Ethyl-4-hydroxy-2-methoxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (83);5-(5-Ethyl-2-hydroxy-4-methoxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (84);5-(5-Ethyl-2,4-dimethoxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (85)

To an argon charged flask containing5-(5-Ethyl-2,4-dihydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (25 mg, 0.055 mmol) andN.N-(Diisopropyl)aminomethylpolystyrene [PS-DIEA] (35 mg, 3.83 mmol/g,2.4 equiv.) was added anhydrous DCM (2.3 ml) and anhydrous methanol(0.25 ml). During gentle stirring, 2M (Trimethylsilyl)diazomethane inhexanes (28 μl, 1.0 equiv.) was added and the solution stirred overnightat room temperature. Argon was bubbled through the solution for 10 mins,the resin filtered off, and the volitiles removed in vacuo. The cruderesidue was purified by semi-preparative HPLC to yield5-(5-Ethyl-4-hydroxy-2-methoxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (83) (5.52 mg, 21%),5-(5-Ethyl-2-hydroxy-4-methoxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (84) (1.14 mg, 4%),5-(5-Ethyl-2,4-dimethoxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (1.46 mg, 5%) and the non-methylated starting material.

83: LCMS (LCT) t_(R)=4.95, MS m/z 466.4 [M+H]⁺

84: LCMS (LCT) t_(R)=5.14, MS m/z 466.4 [M+H]⁺

(85): LCMS (LCT) t_(R)=5.45, MS m/z 480.4 [M+H]⁺

NMR data confirmed the assignments.

Example 86 Ethyl5-(5-chloro-2-hydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxamide

Step 1 Methyl 2-benzoyloxy-5-chloro-benzoate

A mixture of methyl 5-chloro-2-hydroxy-benzoate (2.5 g, 13.4 mmol),K₂CO₃ (3.7 g, 26.8 mmol) and benzyl bromide (2.98 g, 17.4 mmol) inacetone (30 ml) was refluxed for 12 hours. After cooling, acetone wasevaporated. EtOAc (100 ml) was added and filtered. The organic layer wasthen washed with 1M HCl (1×80 ml), brine (2×80 ml) and dried withNa₂SO₄. After filtration and evaporation of the solvent, yellowsemi-solids were obtained (3.2 g). ¹H NMR (d₆-acetone) δ=7.73 (1H, d);7.60-7.30 (1H+5H, m); 7.28 (1H, d); 5.30 (2H, s) and 3.90 (3H, s).

Step 21-(2-Benzyloxy-5-chloro-phenyl)-2-(triphenyl-λ⁵-phosphanylidene)-ethanone

To a stirred suspension of triphenylphosphonium bromide (2.14 g, 6.0mmol) in dried THF (30 ml) at room temperature was added 1.6M n-BuLi inhexane (5.25 ml, 8.39 mmol). The orange suspension was stirred for 3hours. Next, a solution of methyl 2-benzoyloxy-5-chloro-benzoate (0.83g, 3.0 mmol) in THF (8 ml) was slowly added. The resulting mixture wasstirred at 60° C. for 2 hours and filtered after cooled. DCM (100 ml)was added to the filtrate and the combined organic layers were washedwith brine (2×80 ml). After filtration and evaporation of the solvent,yellow oil was obtained (2.0 g). They were then purified bychromatography, eluted with EtOAc:hexane/1:1, yielded 0.97 g solids.R_(f)=0.43. ¹H NMR (d₆-acetone) δ=7.80-7.52 (20H, m); 7.40-7.20(1H+1H+1H, m); 5.25 (2H, s); 4.72 (1H, s, trans-H) and 4.62 (1H, s,cis-H). LCMS: (M+1)⁺=521.2 (RT=5.94 min.)

Step 3 Ethyl 4-(2-benzyloxy-5-chloro-phenyl)-2,4-dioxo-3-(triphenyl-λ⁵phosphanylidene)-butyrate

To a solution of1-(2-Benzyloxy-5-chloro-phenyl)-2-(triphenyl-λ⁵-phosphanylidene)-ethanone(0.49 g, 0.94 mmol), NEt₃ (96 mg, 0.94 mmol) and DMAP (12 mg, 0.09 mmol)in dry toluene (20 ml) at room temperature, ethyl chlorooxoacetate (0.38g, 2.78 mmol) in toluene (5 ml) was added. The mixture was stirred for 2hours and poured into water (50 ml). The organic layer was separated andthe aq. layer was extracted with EtOAc (2×40 ml). The combined organiclayers were then washed with sat. NaHCO₃ solution (2×40 ml), sat. citricacid (1×40 ml), brine (1×40 ml) and dried. Crude oil (0.36 g) waspurified by chromatography, eluted with EtOAc. R_(f)=0.88. ¹H NMR(d₆-acetone) δ=7.75-7.40 (15H, m); 7.30 (1H, dd); 7.15 (1H, d); 7.05(1H, d); 5.10 (2H, s); 3.60 (2H, q) and 1.10 (3H, s). LCMS: (M+1)⁺=621.2(RT=6.49 min.)

Step 4 Ethyl3-(2-benzoyloxy-5-chloro-benzoyl)-3-bromo-3H-azirine-2-carboxylate

To a solution of ethyl4-(2-benzyloxy-5-chloro-phenyl)-2,4-dioxo-3-(triphenyl-λ⁵-phosphanylidene)-butyrate(0.143 g, 0.23 mmol) in DCM (8 ml) at room temperature, a mixture ofTMSN₃ (40 mg, 0.35 mmol) and NBS (62 mg, 0.35 mmol) in DCM (6 ml) wasadded. The resulting solution was stirred for 2 hours. After evaporationof the solvent, the crude product was purified by preparative TLC.Yellow solids (38 mg) were obtained. R_(f)=0.73 (EtOAc:hexane 1:2). ¹HNMR (d₆-acetone) δ=7.80 (1H, d); 7.60 (1H, dd); 7.40 (5H, m); 7.30 (1H,d); 5.20 (2H, s); 4.10 (2H, q) and 1.00 (3H, t). LCMS: (M+1)⁺=438.0(RT=7.32 min.)

Step 5 Ethyl5-(2-benzoyloxy-5-chloro-phenyl)-4-bromo-isoxazole-3-carboxylate

Ethyl 3-(2-benzoyloxy-5-chloro-benzoyl)-3-bromo-3H-azirine-2-carboxylate(55 mg, 0.12 mmol) was heated at reflux in dry toluene for 2 hours.After evaporation of the solvent, crude solids (34 mg) were obtained andpurified by preparative TLC (EtOAc:hexane/1:2). R_(f)=0.73(fluorescent). ¹H NMR (d₆-acetone) δ=7.60 (1H, d); 7.50 (1H, dd); 7.40(1H, d); 7.30 (5H, m); 5.25 (2H, s); 4.42 (2H, q) and 1.40 (3H, t).LCMS: (M+1)⁺=438.0 (RT=7.09 min.)

Step 6 Ethyl5-(2-benzyloxy-5-chloro-phenyl)-4-bromo-isoxazole-3-carboxamide

To a solution of ethyl5-(2-benzoyloxy-5-chloro-phenyl)-4-bromo-isoxazole-3-carboxylate (30 mg,6.8×10^(0.2) mmol) in EtOH (1 ml), ethylamine (70% in water, 1 ml) wasadded. The solution was heated at 100° C. in a CEM® microwave reactor(200 W) for one hour. After that, the solvent was evaporated and thecompound purified by preparative TLC to yield solids (20 mg). R_(f)=0.39(EtOAc:hexane/1:4). ¹H NMR (d₆-acetone) δ=8.10 (1H, s, broad); 7.50 (1H,d); 7.45-7.35 (1H+1H, m); 7.25 (5H, m); 5.20 (2H, s); 3.40 (2H, q) and1.20 (3H, t). LCMS: (M+1)⁺=437.1 (RT=6.57 min.)

Step 7 Ethyl5-(2-benzyloxy-5-chloro-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxamide

A mixture of ethyl5-(2-enzyloxy-5-chloro-phenyl)-4-bromo-isoxazole-3-carboxamide (30 mg,5.6×10⁻² mmol), Pd(Ph₃P)₄ (4 mg, 3.5×10⁻² mmol),4[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]morpholine (63mg, 0.2 mmol) and 1M NaHCO₃ solution (0.2 ml) in DME (1 ml) was stirredat 80° C. under Argon gas for 16 hours. After cooling, the solution wasdiluted with water (8 ml) and extracted with EtOAc (2×20 ml). Thecombined organic layers were washed with brine (1×20 ml) and dried.After filtration and evaporation of the solvents, the crude product waspurified by preparative TLC, yielded 30 mg solids. R_(f)=0.44 (EtOAc).¹H NMR (d₆-acetone) S=8.25 (1H, s, broad); 7.60 (1H, d); 7.55 (1H, dd);7.45 (1H, d); 7.30-6.90 (9H, m); 5.00 (2H, s); 3.55 (4H, m); 3.45(2H+2H, s+q); 2.30 (4H, m) and 1.20 (3H, t). LCMS: (M+1)⁺=532.2 (RT=4.39min.)

Step 8 Ethyl5-(5-chloro-2-hydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxamide

To a solution of ethyl5-(2-benzyloxy-5-chloro-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxamide(25 mg, 4.7×10⁻² mmol) in DCM (5 ml) at 0° C., 1M BCl₃ in DCM (0.15 ml)was added. The resulting cloudy yellow solution was then stirred at 0°C. for 15 minutes and room temperature 3 to 4 hours until it becameclear. After that, the solution was quenched by MeOH (1 ml). Sat. NaHCO₃(1 ml) was then added and extracted with EtOAc (2×2 ml) and dried. Afterthe solvent was filtered and evaporated, the crude oil was purified bypreparative TLC (EtOAc:MeOH/50:1), yielded 12 mg solids. ¹H NMR(d₄-MeOD) δ=7.60 (2H, d); 7.50-7.30 (1H+1H+1H, m); 7.00 (2H, d); 3.70(4H, m); 3.60 (2H, s); 3.50 (2H, q); 2.60 (4H, m) and 1.25 (3H, t).LCMS: (M+1)⁺=442.2 (RT=3.54 min.)

The 4-hydroxy-isomer was prepared in a similar way as its 2-hydroxycounterpart as follows:

Example 87 Ethyl5-(3-chloro-4-hydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxamide

Step 1 Methyl 4-benzoyloxy-3-chloro-benzoate

Methyl 3-chloro-4-hydroxy-benzoate (1.0 g, 5.36 mmol) gave a crude solid(1.57 g). ¹H NMR (d₆-acetone) 8=8.00 (1H, d); 7.95 (1H, dd); 7.60-7.40(5H, m); 7.35 (1H, d); 5.40 (2H, s) and 3.90 (3H, s).

Step 21-(4-Benzyloxy-3-chloro-phenyl)-2-(triphenyl-λ⁵-phosphanylidene)-ethanone

Methyl 4-benzoyloxy-3-chloro-benzoate (1.5 g, 5.40 mmol) gave a crudesolid (2.5 g). R_(f)=0.31 (EtOAc:hexane/1:1). ¹H NMR (d₆-acetone) δ=8.05(1H, d); 7.90 (1H, dd); 7.85-7.35 (20H, m); 7.20 (1H, d); 5.30 (2H, s);4.60 (1H, s, trans-H) and 4.50 (1H, s, cis-H). LCMS: (M+1)⁺=521.2(RT=5.29 min.)

Step 3 Ethyl 4-(4-benzyloxy-3-chloro-phenyl)-2,4-dioxo-3-(triphenyl-λ⁵phosphanylidene)-butyrate

1-(4-Benzyloxy-3-chloro-phenyl)-2-(triphenyl-λ⁵-phosphanylidene)-ethanone(1.84 g, 3.53 mmol) gave a crude solid (1.43 g). ¹H NMR (d₆-acetone)δ=8.00-7.35 (22H, m); 7.20 (1H, d); 5.35 (2H, s); 3.55 (2H, q) and 1.14(3H, s).

LCMS: (M+1)⁺=621.2 (RT=7.29 min.)

Step 4 Ethyl3-(4-benzoyloxy-3-chloro-benzoyl)-3-bromo-3H-azirine-2-carboxylate

Ethyl 4-(4-benzyloxy-3-chloro-phenyl)-2,4-dioxo-3-(triphenyl-λ⁵phosphanylidene)-butyrate (0.74 g, 1.19 mmol) gave a solid (0.168 g)after column and preparative TLC purification. R_(f)=0.24(EtOAc:hexane/1:6). ¹H NMR (d₆-acetone) δ=8.00 (1H, d); 7.90 (1H, dd);7.50 (1H, d); 7.40 (5H, m); 5.40 (2H, s); 4.05 (2H, q) and 0.95 (3H, t).LCMS: (M+1)⁺=438.1 (RT=7.27 min.)

Step 5 Ethyl5-(4-benzoyloxy-3-chloro-phenyl)-4-bromo-isoxazole-3-carboxylate

Ethyl 3-(4-benzoyloxy-3-chloro-benzoyl)-3-bromo-3H-azirine-2-carboxylate(68 mg, 0.16 mmol) gave a solid (20 mg) after preparative TLC andcrystallisation (EtOH). R_(f)=0.26 (fluorescent) (EtOAc:hexane/1:4). ¹HNMR (d₆-acetone) δ=8.00 (1H, d); 7.90 (1H, dd); 7.50 (1H, d); 7.40 (5H,m); 5.35 (2H, s); 4.45 (2H, q) and 1.40 (3H, t). LCMS: (M+1)⁺=438.0(RT=7.39 min.)

Step 6 Ethyl5-(4-benzyloxy-3-chloro-phenyl)-4-bromo-isoxazole-3-carboxamide

Ethyl 5-(4-benzoyloxy-3-chloro-phenyl)-4-bromo-isoxazole-3-carboxylate(10 mg, 2.3×10⁻² mmol) gave a crude solid (8 mg). R_(f)=0.53(EtOAc:hexane/1:2). ¹H NMR (d₆-acetone) δ=8.15 (1H, s, broad); 8.00 (1H,d); 7.90 (1H, dd); 7.50 (1H, d); 7.40 (5H, m); 5.32 (2H, s); 3.42 (2H,q) and 1.20 (3H, t).

Step 7 Ethyl5-(4-benzyloxy-3-chloro-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxamide

Ethyl 5-(4-benzyloxy-3-chloro-phenyl)-4-bromo-isoxazole-3-carboxamide(10 mg, 2.3×10⁻² mmol) gave a crude solid (10 mg), which was then usedin the next step without any further purification.

Step 8 Ethyl5-(3-chloro-4-hydroxy-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxamide

Ethyl5-(4-benzyloxy-3-chloro-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxamide(8 mg, 1.5×10⁻² mmol) gave a crude solid (2 mg) after twice purified bypreparative TLC (EtOAc:MeOH/50:1). ¹H NMR (d₄-MeOD) δ=7.70 (2H, d); 7.60(1H, d); 7.45 (1H+1H, m); 7.00 (2H, d); 3.80 (4H, m); 3.75 (2H, s); 3.50(2H, q); 2.82 (4H, m) and 1.25 (3H, t). LCMS: (M+1)⁺=442.2 (RT=4.47min.)

Example 883-[4-(4-Bromo-phenyl)-isoxazol-5-yl]-5-chloro-2,6-dihydroxy-benzaldehyde

Step 13-(4-Bromo-phenyl)-6-chloro-7-hydroxy-4-oxo-4H-chromene-8-carbaldehyde

3-(4-Bromo-phenyl)-6-chloro-7-hydroxy-chromen-4-one (0.35 g, 1 mmol) andhexamethylene tetramine (0.14 g, 1 mmol) were dissolved in glacialacetic acid (20 ml) and heated overnight at 100° C. Warm 6M HCl (10 ml)was added and the mixture heated for a further hour before being pouredin to water. The precipitate formed was filtered, washed and dried toprovide the pure desired product as a pale brown solid.

LCMS (LCQ) t_(R)=8.27, MS m/z 377.3/379.2 [M−H]⁻

Step 23-[4-(4-Bromo-phenyl)-isoxazol-5-yl]-5-chloro-2,6-dihydroxy-benzaldehyde

To a solution of3-(4-bromo-phenyl)-6-chloro-7-hydroxy-4-oxo-4H-chromene-8-carbaldehyde53.5 mg, 0.14 mmol) in EtOH (6 ml), hydroxylamine hydrochloride (100 mg,1.4 mmol) was added. The resulting mixture was heated at reflux for 16hours. EtOH was evaporated and EtOAc (20 ml) was added. The organiclayer was washed with sat. NaHCO₃ and dried. Solids (33 mg) wereobtained when the resulting oil was triturated with ether. ¹H NMR(d₆-DMSO) δ=9.83 (1H, s); 8.70 (1H, s); 8.21 (1H, s); 7.78 (2H, d) and7.68 (2H, s). LCMS: (M+1)⁺=394.1 (RT=8.60 min.)

Example 895-(5-Ethyl-2-hydroxy-4-methoxy-phenyl)-4-(4-fluoro-phenyl)-isoxazole-3-carboxylicacid hydroxyamide

Step 1 1-(5-Ethyl-2,4-dihydroxy-phenyl)-2-(4-fluoro-phenyl)-ethanone

Ethyl resorcinol (5.37 g, 39 mmol) and 4-fluorophenylacetic acid (6.00g, 39 mmol) were dissolved in etherate BF₃ (40 ml). The solution washeated at 80° C. for 4 hours. When cooled, water (100 ml) was addedcarefully and the solution was extracted with EtOAc (2×80 ml). Theorganic layers were then washed with sat. NaHCO₃ (caution) (2×100 ml)and brine (2×100 ml) and dried with Na₂SO₄. After purification withdecolourising charcoal, a dark green syrup (10.5 g) was obtained.R_(f)=0.4 (EtOAc:n-hexane/1:3). The compound was used in the next stepwithout further purification. ¹H NMR (d₆-acetone) δ=7.80 (1H, s); 7.35(2H, m); 7.00 (1H, m); 6.35 (1H, s); 4.35 (2H, s); 2.55 (2H, q) and 1.10(3H, t).

Step 24-(5-Ethyl-2,4-dihydroxy-phenyl)-3-(4-fluoro-phenyl)-2,4-dioxo-butyricacid ethyl ester

To a solution of1-(5-Ethyl-2,4-dihydroxy-phenyl)-2-(4-fluoro-phenyl)-ethanone (10.3 g,37.6 mmol) in dried pyridine (100 ml) at 0° C., ethyl chlorooxoacetate(15.4 g, 112.8 mmol) was added. The solution was stirred at 0° C. for 4hours and at room temperature for 16 hours. The aq. layer wasneutralised with 1M HCl and extracted with DCM (2×100 ml). The combinedDCM layers were then washed with 2M HCl (2×80 ml), sat. NaHCO₃ (1×100ml), brine (1×100 ml) and dried with Na₂SO₄. After filtration andevaporation of the solvent, dark brown oil was obtained (11.4 g).R_(f)=0.22 (EtOAc:n-hexane/1:2). LCMS shows it is a mixture of desiredproduct [(M−1)⁻=373.1, RT=7.27) and the cyclised chromene carboxylate[(M−1)⁻=355.4, RT=7.83) in a ratio of ca. 6:1. A small amount of samplewas purified by prep. TLC for spectroscopic analysis. ¹H NMR(do-acetone) δ=7.75 (1H, s); 7.30 (2H, m); 7.00 (1H, m); 6.45 (1H, s);4.65 (1H, s); 4.25 (2H, q); 2.55 (2H, q) and 1.10 (6H, t)

Step 36-Ethyl-3-(4-fluoro-phenyl)-7-hydroxy-4-oxo-4H-chromene-2-carboxylicacid ethyl ester

4-(5-Ethyl-2,4-dihydroxy-phenyl)-3-(4-fluoro-phenyl)-2,4-dioxo-butyricacid ethyl ester (3.22 g, 8.6 mmol) was refluxed in a mixture of 0.8MHCl and MeOH (20 ml/20 ml) for 3 hours at 100° C. After that, MeOH wasevaporated and the aq. layer was extracted with EtOAc (2×60 ml). Thecombined organic layers were washed with sat. NaHCO₃ (1×80 ml), brine(2×80 ml), water (1×80 ml) and dried with Na₂SO₄. After purificationwith decolourising charcoal and evaporation of the solvent, brown stickysolids were obtained. They were then extracted with hot ether, darkyellow solids were obtained (0.26 g). R_(f)=0.43 (EtOAc:n-hexane/1:2).LCMS: (M+1)⁺=357.3 (RT=7.83). ¹H NMR (d₆-acetone) δ=9.75 (1H, s); 7.80(1H, s); 7.25 (2H, m); 7.10 (1H, m); 6.90 (1H, s); 4.05 (2H, q); 2.70(2H, q); 1.20 (3H, t) and 0.95 (3H, t).

Step 46-Ethyl-3-(4-fluoro-phenyl)-7-methoxy-4-oxo-4H-chromene-2-carboxylicacid ethyl ester

Iodomethane (0.10 ml, 12 equiv.) was added to a solution of6-Ethyl-3-(4-fluoro-phenyl)-7-hydroxy-4-oxo-4H-chromene-2-carboxylicacid ethyl ester (50 mg, 0.14 mmol) and potassium carbonate (58 mg, 3.0equiv.) in acetone and the mixture refluxed overnight. The volatileswere then evaporated in vacuo and the residue partitioned between water(15 ml) and EtOAc (15 ml). The organic layer was washed with brine,dried over MgSO₄ and evaporated to dryness in vacuo to give a whitecrystalline product (45 mg, 87% yield)

δ_(H) (CDCl₃), 7.96 (1H, s, Ar—H), 7.27 (2H, m, Ar—H), 7.12 (2H, m,Ar—H), 6.92 (1H, s, Ar—H), 4.16 (2H, q, CO₂CH₂CH₃), 3.95 (3H, s, OCH₃),2.71 (3H, q, CH₂CH₃), 1.24 (3H, t, CO₂CH₂CH₃), 1.04 (3H, t, CH₂CH₃)

Step 55-(5-Ethyl-2-hydroxy-4-methoxy-phenyl)-4-(4-fluoro-phenyl)-isoxazole-3-carboxylicacid hydroxyamide

To 6-Ethyl-3-(4-fluoro-phenyl)-7-methoxy-4-oxo-4H-chromene-2-carboxylicacid ethyl ester (25 mg, 0.068 mmol) in ethanol (2.5 ml) was addedhydroxylamine (50% in water, 1 ml) and the solution stirred for 48 h.The volatiles were evaporated off in vacuo and the residue purified bypreparative TLC (10% MeOH in DCM) to give the desired product as a lightbrown solid (3 mg, 12% yield).

LCMS (LCT) t_(R)=6.54, MS m/z 373.17 [M+H]⁺

δ_(H) (d₆-Acetone), 10.73 (1H, broad s), 8.59 (1H, broad s), 7.39 (2H,m, Ar—H), 7.07 (2H, m, Ar—H), 7.00 (1H, s, Ar—H), 6.55 (1H, s, Ar—H),3.82 (3H, s, OCH₃), 2.48 (2H, q, CH₂CH₃), 1.30 (1H, broad s), 1.01 (3H,t, CH₂CH₃).

Example 905-(5-Ethyl-2,4-dihydroxy-phenyl)-4-(4-fluoro-phenyl)-isoxazole-3-carboxylicacid hydroxyamide

To 6-Ethyl-3-(4-fluoro-phenyl)-7-hydroxy-4-oxo-4H-chromene-2-carboxylicacid ethyl ester (25 mg, 0.070 mmol) in ethanol (2.5 ml) was addedhydroxylamine (50% in water, 1 ml) and the solution stirred for 48 hrs.The volitiles were evaporated off in vacuo and the residue purified bypreparative TLC (15% MeOH in DCM) to give the desired product as a brownsolid (2 mg, 8% yield).

LCMS (LCT) t_(R)=5.63, MS m/z 359.13 [M+H]⁺

δ_(H) (d₆-Acetone), 10.72 (1H, broad s, CONH), 8.69 (1H, broad s,Ar—OH), 8.59 (1H, broad s, Ar—OH), 7.39 (2H, m, Ar—H), 7.06 (2H, m,Ar—H), 6.99 (1H, s, Ar—H), 6.52 (1H, s, Ar—H), 2.49 (2H, q, CH₂CH₃),1.31 (1H, broad s), 1.08 (3H, t, CH₂CH₃).

Biological Results

The intrinsic ATPase activity of HSP90 may be measured using yeast HSP90as a model system. The assay, based on the use of malachite green forthe measurement of inorganic phosphate, was used to test the HSP90inhibitory activity of some of the compounds of the Examples herein.

Malachite Green ATPase Assay Materials

Chemicals are of the highest purity commercially available and allaqueous solutions are made up in AR water. Because of the need tominimise contamination with inorganic phosphate, precautions should betaken with solutions and apparatus used in the assays. Glassware and pHmeters are rinsed with double distilled or deionised water before useand, wherever possible, plastic ware should be used. Gloves are worn forall procedures.

-   (1) Greiner 384-well (Greiner 781101) or Costar 384-well    flat-bottomed polystyrene multiwell plates (VWR).-   (2) Assay buffer of (a) 100 mM Tris-HCl, pH 7.4, (b) 150 mM KCl, (c)    6 mM MgCl₂. Stored at room temperature.-   (3) 0.0812% (w/v) malachite green (M 9636, Sigma Aldrich Ltd.,    Poole, UK). Stored at room temperature.-   (4) 2.32% (w/v) polyvinyl alcohol USP (P 1097, Sigma Aldrich Ltd,    Poole, UK) in boiling water (see Comment 1), allowed to cool, and    stored at room temperature.-   (5) 5.72% (w/v) ammonium molybdate in 6 M hydrochloric acid. Stored    at room temperature.-   (6) 34% (w/v) sodium citrate. Stored at room temperature.-   (7) 100 mM ATP, disodium salt, special quality (47699, Sigma    Aldrich). Stored at −20° C.-   (8) E. coli expressed yeast HSP90 protein, purified >95% (see, e.g.,    Panaretou et al., 1998) and stored in 50 uL aliquots at −80° C.

Method

-   -   1. Dilute test compounds to 500 μM in AR water (DMSO        concentration will be 2.5%). Transfer 2.5 μl of these compounds        directly from the daughter plate to the assay plate, giving a        final assay concentration of 100 μM. To obtain 12 point IC50        values, perform serial dilutions 1:2 to produce a range of assay        concentrations from 100 μM to 97.6 nM (2.5% DMSO), and transfer        2.5 μl of each concentration into the assay plate. Column 1 in        the assay plate contains no compound, as a negative control. An        additional row with no compound is also used as a background.    -   2. Prepare ATP by diluting 100 mM stock to 925 μM with assay        buffer, and aliquot 5 μl of diluted ATP to each well including        controls (final assay concentration 370 μM).    -   3. Add 5 μl of buffer to background row.    -   4. Dilute enzyme preparation to 1.05 μM with assay buffer, and        aliquot 5 μl into each compound well and to the negative control        column.    -   5. Collect the reagents to the bottom of the well, cover plate        with plate seal and incubate overnight at 37 deg C.    -   6. First thing in the morning prepare the Malachite Green        Reagent. Add 2 parts of Malachite Green Solution, 1 part of        Polyvinyl Alcohol Solution, 1 part of Ammonium Molybdate        Solution, and 2 parts of AR water.    -   7. Invert to mix, and leave for approximately 1 hour until the        colour turns from brown to golden yellow.    -   8. Add 40 μl of Malachite Green Reagent to each well, allow 5        mins for colour to develop.    -   9. Add 5 μl of Sodium Citrate Reagent to each well (see comment        2)    -   10. Re-cover with plate seal and shake on plate shaker for at        least 15 mins.    -   11. Measure Absorbance at 620 nM using a suitable plate reader        (e.g. Victor, Perkin Elmer Life Sciences, Milton Keynes, UK).        Under these conditions, the control absorbance is 0.9 to 1.4,        and the background is 0.2-0.35 giving a signal to noise ratio of        ˜12. The Z′ factor calculated from data obtained using these        conditions is between 0.6 and 0.9.

Comments

-   (1) The polyvinyl alcohol dissolves in boiling water with difficulty    and stirring for 2-3 h is required.-   (2) The time interval between addition of the malachite green    reagent and the sodium citrate should be kept as short as possible    in order to reduce the non-enzymatic hydrolysis of ATP. Once the    sodium citrate is added, the colour is stable for up to 4 h at room    temperature.-   (3) Compounds can be added to the assay plates using a Biomek FX    Robot (Beckman Coulter). A Multidrop 384 dispenser (Thermo    Labsystems, Basingstoke, UK) can be conveniently used to add    reagents to the plate.-   (4) The assay conditions were optimised with respect to time,    protein and substrate concentration in order to achieve minimal    protein concentration whilst retaining signal to noise differential.-   (5) Signal to noise (S/N) is calculated using the following    equation:

(S−B)/√(SD of S)²+(SD of B)²

-   (6) To determine specific activity of HSP90, a range of inorganic    phosphate concentrations (0-10 μM) are prepared and the absorbance    at 620 nm measured as described. Specific activity is calculated    from the resulting calibration curve.

The compounds tested in the above assay were assigned to one of twoactivity ranges, namely A=<50 μM; B=>50 μM, and those assignments arereported above.

A growth inhibition assay was also employed for the evaluation ofcandidate HSP90 inhibitors:

Assessment of Cytotoxicity by Sulforhodamine B (SRB) Assay: Calculationof 50% Inhibitory Concentration (IC₅₀). Day 1

-   1) Determine cell number by haemocytometer.-   2) Using an 8 channel multipipettor, add 1601 of the cell suspension    (3600 cells/well or 2×10⁴ cells/ml) to each well of a 96-well    microtitre plate.-   3) Incubate overnight at 37° C. in a CO₂ incubator.

Day 2

-   4) Stock solutions of drugs are prepared, and serial dilutions of    each drug are performed in medium to give final concentrations in    wells.-   5) Using a multipipettor, 40 μl of drug (at 5×final concentration)    is added to quadruplicate wells.-   6) Control wells are at either side of the 96 well plates, where 40    μl of medium is added.-   7) Incubate plates in CO₂ incubator for 4 days (48 hours).

Day 6

-   8) Tip off medium into sink and immerse plate slowly into 10% ice    cold trichloroacetic acid (TCA). Leave for about 30 mins on ice.-   9) Wash plates three times in tap water by immersing the plates into    baths of tap water and tipping it off.-   10) Dry in incubator.-   11) Add 100 μl of 0.4% SRB in 1% acetic acid to each well (except    the last row (right hand) of the 96 well plate, this is the 0%    control, ie no drug, no stain. The first row will be the 100%    control with no drug, but with stain). Leave for 15 mins.-   12) Wash off unbound SRB stain with four washes of 1% acetic acid.-   13) Dry plates in incubator.-   14) Solubilise SRB using 100 μl of 10 mM Tris base and put plates on    plate shaker for 5 mins.-   15) Determine absorbance at 540 nm using a plate reader. Calculate    mean absorbance for quadruplicate wells and express as a percentage    of value for control, untreated wells.-   16) Plot % absorbance values versus log drug concentration and    determine the IC₅₀.

By way of illustration, the compound of Example 2 gave an IC50 in the‘A’ range (<50 uM) for the SRB growth arrest assay.

A Fluorescence Polarization_assay was also employed for the evaluationof some of the compounds of the Examples:

Fluorescence Polarization Assay

Fluorescence polarization {also known as fluorescence anisotropy}measures the rotation of a fluorescing species in solution, where thelarger molecule the more polarized the fluorescence emission.

When the fluorophore is excited with polarized light, the emitted lightis also polarized. The molecular size is proportional to thepolarization of the fluorescence emission.

The Fluoroscein-Labelled Probe—RBT0045864-FAM—

binds to HSP90 {full-length human, full-length yeast or N-terminaldomain HSP90} and the anisotropy {rotation of the probe:protein complex}is measured.

Test compound is added to the assay plate, left to equilibrate and theanisotropy measured again. Any change in anisotropy is due tocompetitive binding of compound to HSP90, thereby releasing probe.

Materials

Chemicals are of the highest purity commercially available and allaqueous solutions are made up in AR water.

-   1) Costar 96-well black assay plate #3915-   2) Assay buffer of (a) 100 mM Tris pH7.4; (b) 20 mM KCl; (c) 6 mM    MgCl₂. Stored at room temperature.-   3) BSA (bovine serum albumen) 10 mg/ml (New England Biolabs #    B9001S)-   4) 20 mM probe in 100% DMSO stock concentration. Stored in the dark    at RT. Working concentration is 200 nM diluted in AR water and    stored at 4° C. Final concentration in assay 80 nM.-   5) E. coli expressed human full-length HSP90 protein, purified >95%    (see, e.g., Panaretou et al., 1998) and stored in 50 μL aliquots at    −80° C.

Protocol

-   -   1) Add 100 μl 1× buffer to wells 11A and 12A (=FP BLNK)    -   2) Prepare assay mix—all reagents are kept on ice with a lid on        the bucket as the probe is light-sensitive.

i. Final Conc^(n) 1x Hsp90 FP Buffer  10 ml 1x BSA 10 mg/ml (NEB) 5.0 μl   5 μg/ml Probe 200 μM 4.0 μl  80 nM Human full-length Hsp90 6.25 μl 200 nM

-   -   3) Aliquot 100 μl assay mix to all other wells    -   4) Seal plate and leave in dark at room temp for 20 minutes to        equilibrate

Compound Dilution Plate—1×3 Dilution Series

-   -   1) In a clear 96-well v-bottom plate—{# VWR 007/008/257} add 10        μl 100% DMSO to wells B1 to H11    -   2) To wells A1 to A11 add 17.5 μl 100% DMSO    -   3) Add 2.5 μl cpd to A1. This gives 2.5 mM {50×} stock        cpd—assuming cpds 20 mM.    -   4) Repeat for wells A2 to A10. Control in columns 11 and 12.    -   5) Transfer 5 μl from row A to row B— not column 12. Mix well.    -   6) Transfer 5 μl from row B to row C. Mix well.    -   7) Repeat to row G.    -   8) Do not add any compound to row H—this is the 0 row.    -   9) This produces a 1×3 dilution series from 50 μM to 0.07 μM.    -   10) In well B12 prepare 20 μl of 100 μM standard compound.    -   11) After first incubation the assay plate is read on a Fusion™        a-FP plate reader (Packard BioScience, Pangbourne, Berkshire,        UK).    -   12) After the first read, 2 μl of diluted compound is added to        each well for columns 1 to 10. In column 11 {provides standard        curve} only add compound B11-H11. Add 2 μl of 100 mM standard        cpd to wells B12-H12 {is positive control}    -   13) The Z′ factor is calculated from zero controls and positive        wells. It typically gives a value of 0.7-0.9.

The compounds tested in the above assay were assigned to one of twoactivity ranges, namely A=<10 μM; B=>10 μM, and those assignments arereported above. By way of illustration, the compound of Example 2 gavean IC50 in the ‘A’ range.

REFERENCES

A number of publications are cited above in order to more fully describeand disclose the invention and the state of the art to which theinvention pertains. Full citations for these references are providedbelow. Each of these references is incorporated herein by reference inits entirety into the present disclosure.

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1. A method of treatment of diseases or conditions responsive toinhibition of HSP90 activity in mammals comprising administering to themammal an amount of a compound of formula (A) or (B) or a salt, N-oxide,hydrate or solvate thereof, or a prodrug thereof effective to inhibitHSP90 activity:

wherein R₁ is a group of formula (IB)

wherein in any compatible combination R represents one or more optionalsubstituents, Alk¹ and Alk² are optionally substituted divalent C₁-C₆alkylene or C₂-C₆ alkenylene radicals, p, r and s are independently 0 or1, Z is —O—, —S—, —(C═O)—, —(C═S)—, —SO₂—, —C(═O)O—, —C(═O)NR^(A)—,—C(═S)NR^(A)—, —SO₂NR^(A)—, —NR^(A)C(═O)—, —NR^(A)SO₂— or —NR^(A)—wherein R^(A) is hydrogen or C₁-C₆ alkyl, and Q is hydrogen or anoptionally substituted carbocyclic or heterocyclic radical; R₂ is (i) agroup of formula (IA):—Ar¹-(Alk¹)_(p)-(Z)_(r)-(Alk²)_(s)-Q  (IA) wherein in any compatiblecombination Ar¹ is an optionally substituted aryl or heteroaryl radical,and Alk¹, Alk², p, r, s, Z, R^(A) and Q are as defined in relation toR₁; (ii) a carboxamide radical; or (iii) a non aromatic carbocyclic orheterocyclic ring wherein a ring carbon is optionally substituted,and/or a ring nitrogen is optionally substituted by a group of formula-(Alk¹)_(p)-(Z)_(r)-(Alk²)_(s)-Q wherein Q, Alk¹, Alk², Z, p, r and sare as defined above in relation to group (IA); and R₃ is hydrogen,optionally substituted cycloalkyl, cycloalkenyl, C₁-C₆ alkyl, C₁-C₆alkenyl, or C₁-C₆ alkynyl; or a carboxyl, carboxamide, or carboxyl estergroup. 2-70. (canceled)